erbB-3 nucleic acids

ABSTRACT

A DNA fragment distinct from the epidermal growth factor receptor (EGFR) and erbB-2 genes was detected by reduced stringency hybridization of v-erbB to normal genomic human DNA. Characterization of the cloned DNA fragment mapped the region of v-erbB homology to three exons with closest homology of 64% and 67% to a contiguous region within the tyrosine kinase domains of the EGFR and erbB-2 proteins, respectively. cDNA cloning revealed a predicted 148 kd transmembrane polypeptide with structural features identifying it as a member of the erbB family, prompting designation of the new gene as erbB-3. It was mapped to human chromosome 12q11-13 and was shown to be expressed as a 6.2 kb transcript in a variety of normal tissues of epithelial origin. Markedly elevated erbB-3 mRNA levels were demonstrated in certain human mammary tumor cell lines. These findings indicate that increased erbB-3 expression, as in the case of EGFR and erbB-2, plays a role in some human malignancies. Using erbB-3 specific antibodies (polyclonal or monoclonal), the erbB-3 protein was identified as a 180 kDa glycoprotein, gp180 erbB-3 .

This application is a divisional of, and claims the benefit of,application Ser. No. 08/473,119, filed Jun. 7, 1995, now U.S. Pat. No.5,820,859, which is a divisional of Ser. No. 07/978,895, filed Nov. 10,1992, now U.S. Pat. No. 5,480,968, which is a continuation-in-part ofSer. No. 07/444,406, filed Dec. 1, 1989, now U.S. Pat. No. 5,183,884which applications are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to genes which encode novel proteinsrelated to a family of receptor proteins typified by two relatedmembrane spanning tyrosine kinases: the Epidermal Growth Factor receptor(EGFR), which is encoded by the erbB gene, the normal human counterpartof an oncogene (v-erbB) that was first recognized in the proviral DNA ofavian erythroblastosis virus; and the receptor encoded by the relatedgene erbB-2. In particular, the present invention relates to a DNAsegment encoding the coding sequence, or a unique portion thereof, for athird member of this receptor gene family, herein designated erbB-3.

BACKGROUND OF THE INVENTION

Proto-oncogenes encoding growth factor receptors constitute severaldistinct families with close overall structural homology. The highestdegree of homology is observed in their catalytic domains, essential forthe intrinsic tyrosine kinase activity of these proteins. Examples ofsuch receptor families include: the EGFR and the related product of theerbB-2 oncogene; the Colony Stimulating Factor 1 receptor (CSF-1-R) andthe related Platelet-Derived Growth Factor receptor (PDGF-R); theinsulin receptor (IR) and the related Insulin-like Growth factor 1receptor (IGF-1R); and the receptors encoded by the related oncogeneseph and elk.

It is well established that growth factor receptors in several of thesefamilies play critical roles in regulation of normal growth anddevelopment. Recent studies in Drosophila have emphasized how criticaland multifunctional are developmental processes mediated byligand-receptor interactions. An increasing number of Drosophila mutantswith often varying phenotypes have now been identified as being due tolesions in genes encoding such proteins. The genetic locus of theDrosophila EGFR homologue, designated DER, has recently been identifiedas being alilelic to the zygotic embryonic lethal faint little ballexhibiting a complex phenotype with deterioration of multiple tissuecomponents of ectodermal origin. Furthermore, other mutants appear tolack DER function either in the egg or the surrounding maternal tissue.Thus, the DER receptor may play an important role in the ligand-receptorinteraction between egg and follicle cells necessary for determinationof correct shape of eggshell and embryo. It is not yet known whether DERrepresents the sole Drosophila counterpart of known mammalianerbB-related genes.

Some of these receptor molecules have been implicated in the neoplasticprocess as well. In particular, both the erbB and erbB-2 genes have beenshown to be activated as oncogenes by mechanisms involvingoverexpression or mutations that constitutively activate the catalyticactivity of their encoded receptor proteins (Bargmann, C. I., Hung, M.C. & Weinberg, R. A., 1986, Cell 45:649-657; Di Fiore, P. P., Pierce, J.H., Kraus, M. H., Segatto, O., King, C. R. & Aaronson, S. A., 1987,Science 237:178-182; Di Fiore, P. P., Pierce, J. H., Fleming, T. P.,Hazan, R., Ullrich, A., King, C. R., Schlessinger, J. & Aaronson, S. A.,1987, Cell 51:1063-1070; Velu, T. J., Beguinot, L., Vass, W. C.,Willingham, M. C., Merlino, G. T., Pastan, I. & Lowy, D. R., 1987,Science 238:1408-1410). Both erbB and erbB-2 have been causallyimplicated in human malignancy. erbB gene amplification oroverexpression, or a combination of both, has been demonstrated insquamous cell carcinomas and glioblastomas (Libermann, T. A., Nusbaum,H. R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield,M. D., Ullrich, A. & Schlessinger, J., 1985, Nature 313:144-147). erbB-2amplification and overexpression have been observed in human breast andovarian carcinomas (King, C. R., Kraus, M. H. & Aaronson, S. A, 1985,Science 229:974-976; Slamon, D. J., Godolphin, W., Jones, L. A., Holt,J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove,J., Ullrich, A. & Press, M. F., 1989, Science 244:707-712), and erbB-2overexpression has been reported to be an important prognostic indicatorof particularly aggressive tumors (Slamon, D. J., et al., 1989, supra).Yet, not all such tumors have been found to overexpress erbB-2, and manyhuman tumors have not yet been associated with any known oncogene. Thus,there has been a continuing need to search for additional oncogeneswhich would provide knowledge and methods for diagnosis and, ultimately,for rational molecular therapy of human cancers.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

It is an object of present invention to provide a DNA segment encoding areceptor protein related to the erbB proto-oncogene family whichpreviously has not been known or even suspected to exist. Further, it isan object of the present invention to develop assays for expression ofthe RNA and protein products of such genes to enable determining whetherabnormal expression of such genes is involved in human cancers. Thus,further objects of this invention include providing antibodies, eitherpolyclonal or monoclonal, specific to a unique portion of the receptorprotein; a method for detecting the presence of an erbB-3 ligand that iscapable of either activating or down-regulating the receptor protein aswell as procedures for purifying the resultant ligand; a method ofscreening potential ligand analogs for their ability to activate thereceptor protein; and procedures for targeting a therapeutic drug tocells having a high level of the receptor protein.

In pursuit of the above objects, the present inventors have discovered ahuman genomic DNA fragment that is produced by cleavage with the SacIrestriction enzyme, has a size of about 9 kbp, and is detectable bynucleic acid hybridization with a probe derived from the v-erbB geneonly under reduced stringency hybridization conditions. Thus, this DNAfragment is distinct from those known to encode the epidermal growthfactor receptor (EGFR) (i.e., the erbB gene) and from the related erbB-2gene. Characterization of this DNA fragment after partial purificationand molecular cloning showed that the region of v-erbB homology mappedto three exons that encode amino acid sequences having homologies of 64%and 67% to contiguous regions within the tyrosine kinase domains of theEGFR and erbB-2 proteins, respectively. A probe derived from the genomicDNA clone identified cDNA clones of the related mRNA which encode apredicted 148 kd transmembrane polypeptide with structural featuresidentifying it as a member of the erbB family, prompting designation ofthe new gene as erbB-3. This gene was mapped to human chromosome12q11-13 and was shown to be expressed as a 6.2 kb transcript in avariety of normal tissues of epithelial origin. Markedly elevated erbB-3mRNA levels were demonstrated in certain human mammary tumor cell lines.

The predicted human erbB-3 gene product is closely related to EGFR anderbB-2, which have been implicated as oncogenes in model systems andhuman neoplasia. The erbB-3 coding sequence was expressed in NIH/3T3fibroblasts and its product was identified as a 180 kDa glycoprotein,gp180^(erbB-3). Tunicamycin and pulse-chase experiments revealed thatthe mature protein was processed by N-linked glycosylation of a 145 kDaerbB-3 core polypeptide. The intrinsic catalytic function ofgp180^(erbB-3) was uncovered by its ability to autophosphorylate invitro. Ligand-dependent signaling of its cytoplasmic domain wasestablished employing transfectants which express a chimeric EGFR/erbB-3protein, gp180^(EGFR/erbB-3). EGF induced tyrosine phosphorylation ofthe chimera and promoted soft agar colony formation of suchtransfectants. These findings, combined with the detection ofconstitutive tyrosine phosphorylation of gp180^(erbB-3) in 4 out of 12human mammary tumor cell lines, implicates the activated erbB-3 productin the pathogenesis of some human malignancies.

Accordingly, in a principal embodiment, the present invention relates toa DNA segment having a nucleotide sequence that encodes an erbB-3 geneor a unique portion thereof. This portion of an erbB-3 gene includes atleast about 12 to 14 nucleotides which are sufficient to allow formationof a stable duplex with a DNA or RNA segment having sequencescomplementary to those in this portion of an erbB-3 gene. Further, thisunique portion of an erbB-3 gene, of course, has a sequence not presentin an erbB or an erbB-2 gene. In other words, the sequence of thisportion of an erbB-3 gene differs in at least one nucleotide from thesequence of any other DNA segment. In one embodiment, this DNA segmentis exemplified by a human genomic DNA fragment that is produced bycleavage with the SacI restriction enzyme, has a size of about 9 kbp,and is detectable by nucleic acid hybridization with a probe derivedfrom the v-erbB gene only under reduced stringency hybridizationconditions, as described in Example 1. By application of the nucleicacid hybridization and cloning methods described in the presentdisclosure, without undue experimentation, one of ordinary skill in theart of recombinant DNA is enabled to identify and isolate DNA fragmentsrelated to the present human DNA fragment comprising a nucleotidesequence that encodes at least a portion of a mammalian erbB-3 geneother than the human erbB-3 gene. Application of the genomic DNAfragment of the erbB-3 gene as a probe in hybridization methods alsoenables one of ordinary skill in the art to obtain an entire erbB-3gene, by sequential isolation of overlapping fragments adjoining thepresent fragment, i.e., by an approach known in the art as chromosomewalking.

The present disclosure describes the partial nucleotide sequence of thehuman genomic 9 kbp SacI DNA fragment, within the region of homology tothe v-erbB gene; however, the methods in the present disclosure furtherenable the isolation and determination of the sequence of the entire 9kbp human genomic DNA fragment according to the present invention.Accordingly, the present invention further relates to a DNA segmenthaving the nucleotide sequence, or a unique portion thereof, of a humangenomic DNA fragment that is produced by cleavage with the SacIrestriction enzyme, has a size of about 9 kbp, and is detectable bynucleic acid hybridization with a probe derived from the v-erbB geneonly under reduced stringency hybridization conditions, as described inExample 1. By extension of the chromosome walking approach noted above,the present invention further enables one of ordinary skill in the artto determine the sequences of related DNA fragments comprising thecomplete human erbB-3 gene as well as erbB-3 genes of, for example,mammals other than human.

In the application of the present SacI DNA fragment or any portionthereof as a probe for nucleic acid hybridization, the fragment isamplified, for example, by the in vitro polymerase chain reaction method(PCR; see U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,683,195; and Saiki etal., 1985, Science 230:1350-54) or by standard methods of molecularcloning. For example, a clone of the human erbB-3 gene DNA segmentaccording to the present invention is exemplified by a recombinant cloneof a normal human thymus DNA fragment, herein designated as the E3-1gcnomic clone, having the partial restriction enzyme map defined in FIG.2 and the partial DNA sequence defined in FIG. 3 and SEQ ID NO:1 of thepresent application. Isolation and characterization of genomic cloneE3-1 is described in Example 2, below.

Analysis of the nucleotide sequences of the human genomic DNA segmentaccording to the present invention reveals that the nucleotide sequenceencodes three open reading frames bordered by splice junction consensussequences which define the boundaries between nontranslated intronsequences and the translated exons (shown in FIG. 2 and SEQ ID NO:1).The predicted amino acid sequences of the three exons (SEQ ID NOS:1 and2) are highly similar to three regions which are contiguous in thetyrosine kinase domains of v-erbB, as well as human EGFR and erbB-2proteins. Moreover, the predicted amino acid sequences of this humangenomic clone are included in a larger open reading frame incomplementary DNA (cDNA) clones of an mRNA species that is detected byhybridization of a probe derived from the human genomic DNA clone.

Accordingly, the present invention also relates to a DNA segment havinga nucleotide sequence of an erbB-3 gene in which that nucleotidesequence encodes the amino acid sequence of an erbB-3 protein or aunique portion thereof. In other words, the sequence of this portion ofan erbB-3 amino acid sequence differs in at least one amino acid residuefrom the amino acid sequence encoded by any other DNA segment. Thisportion of an erbB-3 amino acid sequence includes at least about 4 to 6amino acids which are sufficient to provide a binding site for anantibody specific for this portion of the erbB-3 polypeptide. Further,this unique portion of an erbB-3 amino acid sequence, of course,includes sequences not present in an erbB or an erbB-2 gene. Inparticular, the present invention relates to such a DNA segment forwhich this amino acid sequence or unique portion thereof is that of thepolypeptide product of the human erbB-3 gene. This DNA segment isexemplified by the human genomic DNA clone E3-1, above, as well as byhuman cDNA cones designated E3-6, E3-8, E3-9, E3-11 and E3-16, which aredescribed in Example 3 below. A preferred embodiment of this DNA segmentthat encodes the amino acid sequence of the entire polypeptide productof the human erbB-3 gene is human cDNA clone E3-16 having the nucleotidesequence defined in SEQ ID NO:3 and having the predicted amino acidsequence defined in SEQ ID NOS:3 and 4.

The DNA segments according to this invention are useful for detection ofexpression of erbB-3 genes in normal and tumor tissues, as described inExample 5 below. Therefore, in yet another aspect, the present inventionrelates to a bioassay for determining the amount of erbB-3 mRNA in abiological sample comprising the steps of: i) contacting that biologicalsample with a nucleic acid isolate consisting essentially of anucleotide sequence that encodes erbB-3 or a unique portion thereofunder conditions such that a nucleic acid:RNA hybrid molecule, such as aDNA:RNA hybrid molecule, can be formed; and ii) determining the amountof hybrid molecule present, the amount of hybrid molecule indicating theamount of erbB-3 mRNA in the sample. Findings described in Example 5,below, indicate that increased erbB-3 expression, as detected by thismethod of this invention, plays a role in some human malignancies, as isthe case for the EGFR (erbB) and erbB-2 genes.

Of course, it will be understood by one skilled in the art of geneticengineering that in relation to production of erbB-3 polypeptideproducts, the present invention also includes DNA segments having DNAsequences other than those in the present examples that also encode theamino acid sequence of the polypeptide product of an erbB-3 gene. Forexample, it is known that by reference to the universal genetic code,standard genetic engineering methods can be used to produce syntheticDNA segments having various sequences that encode any given amino acidsequence. Such synthetic DNA segments encoding at least a portion of theamino acid sequence of the polypeptide product of the human erbB-3 genealso fall within the scope of the present invention. Further, it isknown that different individuals may have slightly different DNAsequences for any given human gene and, in some cases, such mutant orvariant genes encode polypeptide products having amino acid sequenceswhich differ among individuals without affecting the essential functionof the polypeptide product. Still further, it is also known that manyamino acid substitutions can be made in a polypeptide product by geneticengineering methods without affecting the essential function of thatpolypeptide. Accordingly, the present invention further relates to a DNAsegment having a nucleotide sequence that encodes an amino acid sequencediffering in at least one amino acid from the amino acid sequence ofhuman erbB-3, or a unique portion thereof, and having greater overallsimilarity to the amino acid sequence of human erbB-3 than to that ofany other polypeptide. The amino acid sequence of this DNA segmentincludes at least about 4 to 6 amino acids which are sufficient toprovide a binding site for an antibody specific for the portion of apolypeptide containing this sequence. In a preferred embodiment this DNAsegment encodes an amino acid sequence having substantially the functionof the human erbB-3 polypeptide. As noted above, the predicted erbB-3polypeptide is a 148 kd transmembrane polypeptide with structuralfeatures identifying it as a member of the erbB receptor family.

The similarity of the amino acid sequence of the present invention withthat of an erbB-3 amino acid sequence is determined by the method ofanalysis defined by the sequence alignment and comparison algorithmsdescribed by Pearson and Lipman (Pearson, W. R. & Lipman, D. J., 1988,Proc. Nat. Acad. Sci. U.S.A. 85:2444-48). This comparison contemplatesnot only precise homology of amino acid sequences, but alsosubstitutions of one residue for another which are known to occurfrequently in families of evolutionarily related proteins sharing aconserved function.

The present invention further relates to a recombinant DNA moleculecomprising a DNA segment of this invention and a vector. In yet anotheraspect, the present invention relates to a culture of cells transformedwith a DNA segment according to this invention. These host cellstransformed with DNAs of the invention include both higher eukaryotes,including animal, plant and insect cells, and lower eukaryotes, such asyeast cells, as well as prokaryotic hosts including bacterial cells suchas those of E. coli and Bacillus subtilis. These aspects of theinvention are exemplified by recombinant DNAs and cells described inExamples 2, 3 and 6, below.

One particular embodiment of this aspect of this invention comprises acell, preferably a mammalian cell, transformed with a DNA of theinvention, wherein the transforming DNA is capable of being expressed toproduce the functional polypeptide of an erbB-3 gene. For example,mammalian cells (COS-1) transformed with the pSV2 gpt vector carryingthe E3-16 cDNA are prepared according to well-known methods, such asthose described in U.S. patent application Ser. No. 07/308,302 of Matsuiet al., filed Feb. 9, 1989; see also Pierce, J. H. et al., 1988, Science239:628-631; and Matsui, T., Heidaran, M., Miki, T., Popescu, N., LaRochelle, W., Kraus, M., Pierce, J. & Aaronson, S., 1989 Science243:800-804). Briefly, cDNA expression plasmids are constructed byintroducing the erbB-3-related cDNA encompassing all the nucleotides inthe open reading frame into the pSV2 gpt vector into which the simiansarcoma virus long-terminal-repeat (LTR) had been engineered as thepromotor, as previously described in detail. Transient expression of theerbB-3 gene in such recombinant vectors is achieved by transfection intoCOS-1 cells.

Stable expression of an erbB-3 gene can also be obtained with mammalianexpression vectors such as the pZIPNEOSVX vector (Cepko, C. L., RobertsB. E. and Mulligan, R. C., 1984, Cell 37:1053-62). For example, aeukaryotic expression vector was engineered by cloning the full-lengtherbB-3 coding sequence derived from cDNA clone E3-16 into the BamHI siteof the pZIPNEOSVX vector DNA adapting the DNA fragments with syntheticoligonucleotides. NIH/3T3 cells were transfected with 1 μg ofrecombinant expression vector DNA (LTRerbB-3) and selected with theresistance marker antibiotic G418. To detect expression of erbB-3,polyclonal rabbit antiserum was raised against a synthetic peptide (suchas amino acid (aa) positions 1191-1205 (SEQ ID NO:5); aa 1254-1268 (SEQID NO:6); aa 478-492 (SEQ ID NO:7); aa 1116-1130 (SEQ ID NO:8) and aa1199-1213 (SEQ ID NO:9)). These peptide epitopes are locatedintracellularly within the predicted carboxyl terminus of the erbB-3coding sequence with the exception of aa 478-492, which resides in theextracellular domain of the erbB-3 protein. For example, as shown inFIG. 7, immunoblotting analysis using antiserum raised against aa1191-1205 led to detection of the erbB-3 protein (panel A). Thespecificity of erbB-3 protein detection was demonstrated bypreincubating the antiserum with the homologous peptide (panel B).Moreover, the normal 180 kD erbB-3 protein was specifically detectedwith the polyclonal antiserum only in cells transfected with therecombinant erbB-3 expression vector, while control NIH3T3 cells thatwere not transfected with the vector were negative. There was nocross-reactivity of the above-listed antisera with the related EGFR orerbB-2 proteins overexpressed in NIH/3T3 cells. The stably transfectedNIH3T3 cells are useful as erbB-3 receptor protein sources for testingpotential candidates for an erbB-3-specific ligand, analysis of thebiological activity, as well as generation of monoclonal antibodiesraised against the native erbB-3 protein. An erbB-3-specific ligand isidentified by detection of autophosphorylation of the erbB-3 receptorprotein, stimulation of DNA synthesis or induction of the transformedphenotype of the LTRerbB-3 transfected NIH3T3 cells.

Alternatively, other transformed cell systems are available forfunctional expression of receptors of the erbB receptor family, forexample, a system based on the 32D cell line, a mouse hematopoietic cellline normally dependent on interleukin-3 (I1-3) for survival andproliferation. Recent studies have established that introduction of anexpression vector for the EGFR in these cells leads to effectivecoupling with EGF mitogenic signal transduction pathways, therebyallowing a ligand of the EGFR to replace I1-3 in supporting survival andgrowth of the 32D cells. By employing the known methods described forthe EGFR, for example (Pierce, J. H. et al., 1988, supra), the E3-16cDNA of the present invention is expressed to produce functionalreceptors in 32D cells which are then useful for examining thebiological function of these erbB-3 receptors, for instance, thespecificity of their ligand binding capacity and coupling capacities tosecondary messenger systems. Thus, by so using gene expression methodsdescribed herein with the DNAs of the present invention, especially thepreferred E3-16 cDNA clone, one of ordinary skill in the art, withoutundue experimentation, can construct cell systems which fall within thescope of this invention, for determining the mechanisms of erbB-3regulatory processes. Accordingly, the present invention also relates toa bioassay for screening potential analogs of ligands of erbB-3receptors for the ability to affect an activity mediated by erbB-3receptors, comprising the steps of: i) contacting a molecule suspectedof being a ligand with erbB-3 receptors produced by a cell that yieldsfunctional erbB-3 receptors; ii) determining the amount of a biologicalactivity mediated by those erbB-3 receptors; and iii) selecting thoseanalogs which affect the biological activity mediated by the erbB-3receptors. For example, a compound can be added to a cell having normalor low level erbB-3 phosphorylation. The amount of erbB-3phosphorylation is then measured and compared to the level prior toadding the compound. The presence of increased activity can then beselected. Alternatively, a cell with high or constitutive erbB-3phosphorylation can be used to screen for compounds which decreaseactivity. In addition, an erbB-3 ligand or analogs can be used in thissystem to screen for the amount of ligand which is necessary to promoteor inhibit phosphorylation.

Various standard recombinant systems, such as those cited above as wellas others known in the art, are suitable as well for production of largeamounts of the novel erbB-3 receptor protein using methods of isolationfor receptor proteins that are well known in the art. Therefore, thepresent invention also encompasses an isolated polypeptide having atleast a portion of the amino acid sequence defined in FIG. 4 (SEQ IDNO:4), such as those polypeptides given by SEQ ID NOS:5-9.

The invention further presents results undertaken in an effort toidentify and characterize the normal erbB-3 gene product (Examples 6-8).By analysis of an EGFR/erbB-3 chimeric receptor, this inventiondemonstrates that EGF-dependent activation of the erbB-3 catalyticdomain results in a proliferative response in transfected NIH/3T3 cells.Further, the invention shows that some human mammary tumor cell linesexhibit a dramatic elevation of steady state erbB-3 tyrosinephosphorylation, implying functional erbB-3 activation in these tumorcells.

The identification of erbB-3 ligands is of great importance because, forinstance, the availability of these ligands will facilitate the completecharacterization of erbB-3 biological function as well as development oftherapeutic strategies involving the ligands. In particular, the instantobservation of functional erbB-3 activation in mammary tumor cells atsteady state raises the possibility that a role of erbB-3 in humantumors involves autocrine activation. That is, the simultaneousexpression of the ligand by the tumor cell may constitutively activateerbB-3, leading to an uncontrolled proliferative growth response.Accordingly, this invention provides for the detection, purification andcharacterization of erbB-3 ligands, particularly erbB-3 ligands that arecapable of either activating or down-regulating (blocking the activationof) the erbB-3 protein.

The ligand detection and purification method of this inventioncapitalizes on the erbB-3 expression and activation characteristics incertain cell lines as well as the common property of growth factorreceptor tyrosine kinases to rapidly autophosphorylate on tyrosineresidues in response to ligand triggering to detect activating orblocking ligand from source containing potential erbB-3 ligands, asdescribed in Example 9. Therefore, in yet another aspect, the presentinvention relates to a method for detecting the presence of an erbB-3ligand in a source containing a potential erbB-3 ligand, comprising thesteps of a) contacting a first sample of cells from a cell line thatexpresses erbB-3 protein with the source containing a potential erbB-3ligand for a time and under conditions sufficient to allow erbB-3 ligandcontained in the source to bind to erbB-3 protein to form a triggeredsample, wherein the cell line expresses erbB-3 protein having low levelintrinsic tyrosine phosphorylation; b) contacting a second sample ofcells from the cell line with a control medium (unconditioned serum freemedium) for the time and under the conditions as given in step a) aboveto form a control sample; c) determining the level of erbB-3 activationin the triggered sample and in the control sample; and d) comparing thelevel of erbB-3 activation in the triggered sample with the level oferbB-3 activation in the control sample, wherein an increase inactivation in the triggered sample over the control sample indicates thepresence of an erbB-3 activating ligand in the source containing apotential erbB-3 ligand. Alternatively, chimeric receptors, as shown inFIG. 11, can be utilized to screen for erbB-3 ligands. The erbB-3activation can be ascertained by measuring the level of erbB-3 tyrosinephosphorylation in the triggered sample and in the control sample (anincrease in the level of erbB-3 tyrosine phosphorylation correlates withan increase in the level of erbB-3 protein activation); measuring thelevel of cell growth in the triggered sample and in the control sample(wherein an increase in the level of cell growth correlates with anincrease in the level of erbB-3 activation) or measuring the level ofDNA synthesis for the cells in the triggered sample and in the controlsample (an increase in the level of DNA synthesis for the cellscorrelates with an increase in the level of erbB-3 activation).

Similarly, the presence of an erbB-3 blocking or inhibiting ligand in asource containing a potential erbB-3 ligand can be detected by a)contacting a first sample of a cell line that expresses erbB-3 proteinwith the source containing a potential erbB-3 ligand for a time andunder conditions sufficient to allow erbB-3 ligand contained in thesource to bind to erbB-3 protein to form a blocked sample, wherein thecell line expresses erbB-3 protein having high level intrinsic tyrosinephosphorylation; b) contacting a second sample of the cell line with acontrol medium for the time and under the conditions as given in step a)to form a control sample; c) determining the level of erbB-3 activationin the blocked sample and in the control sample; and d) comparing thelevel of erbB-3 activation in the blocked sample with the level oferbB-3 activation in the control sample, wherein a decrease inactivation in the blocked sample over the control sample indicates thepresence of an erbB-3 blocking ligand in the source containing apotential erbB-3 ligand. Alternatively, chimaric receptors, as shown inFIG. 11, can be utilized to screen for erbB-3 blocking ligands.

In addition, the concentration of various ligands can be utilized toaffect the erbB-3 activity. For example, a ligand which promotes erbB-3activity at low concentrations can be administered or promoted to highconcentrations which can inhibit erbB-3 activity.

This invention additionally provides a method of decreasing abiochemical or biological activity mediated by the erbB-3 receptor,comprising blocking the binding of an erbB-3 activating ligand with theerbB-3 receptor. The blocking can be accomplished by an antibodyreactive with the ligand binding domain of the erbB-3 receptor or by anerbB-3 blocking ligand. Furthermore, a method of promoting a biochemicalor biological activity mediated by the erbB-3 receptor, comprisingcontacting an erbB-3 activating ligand with the erbB-3 receptor isprovided.

This invention also provides a method of detecting the overexpression oferbB-3 in a sample from a subject. The method comprises detecting theamount of erbB-3 in the sample and comparing the amount in the sample tothe amount in an equivalent sample having normal expression, thepresence of erbB-3 in a greater amount indicating overexpression oferbB-3. By “greater amount” is meant a statistically significant amount.Such amount depends on the conditions utilized and can readily bedetermined given the teachings set forth herein. Generally, a two-foldor greater increase would be predictive of overexpression. erbB-3 can bedetected, for example, by detecting mRNA utilizing Northernhybridization, RNA dot blot, RNA slot blot, or in situ hybridization.erbB-3 can also be detected at the protein level utilizing, for example,Western blots, immunoprecipitation, immunohistochemistry, ELISA, andradioimmunoassay. Once overexpression is detected, the overexpression oferbB-3 can be correlated to a tumor. Such correlation can be used todiagnose a tumor or monitor the progression of a previously diagnosedtumor.

Also provided is a method of detecting the activation of erbB-3 in atest sample from a subject, comprising detecting the presence ofphosphorylation of erbB-3, the presence of phosphorylation of erbB-3indicating the presence of erbB-3 activation in the sample. This methodcan further comprise comparing the amount of erbB-3 phosphorylation inthe test sample to the amount of erbB-3 phosphorylation in a sample froma normal subject and correlating an increase in phosphorylation in thetest sample, with the presence of a neoplastic condition in the subject.Such correlation can be used to diagnose a tumor or monitor theprogression of a previously diagnosed tumor.

This invention further comprises a purified antibody specific for thehuman erbB-3 polypeptide having the amino acid sequence defined in FIG.4 (SEQ ID NO:4) or the mature gp180^(erbB-3) protein or a unique portionthereof, such as those polypeptides given by SEQ ID NOS:5-9. In thisembodiment of the invention, the antibodies are monoclonal or polyclonalin origin, and are generated using erbB-3 receptor-related polypeptidesor peptides from natural, recombinant or synthetic chemistry sources.The term “specific” refers to an erbB-3 antibody capable of binding orotherwise associating nonrandomly with an antigen of erbB-3 such that itdoes not cross react substantially with other antigens. These antibodiesspecifically bind to an erbB-3 protein which includes the sequence ofsuch polypeptide. In other words, these antibodies bind substantiallyonly to erbB-3 receptor proteins and not to erbB (EGFR) or erbB-2proteins. Also, preferred antibodies of this invention bind to an erbB-3protein when that protein is in its native (biologically active)conformation. For instance, MAb E-31 has been shown to detect the nativeerbB-3 protein.

Fragments of antibodies of this invention, such as Fab or F(ab)′fragments, which retain antigen binding activity and can be prepared bymethods well known in the art, also fall within the scope of the presentinvention. Further, this invention comprises a pharmaceuticalcomposition of the antibodies of this invention, or an active fragmentthereof, which can be prepared using materials and methods for preparingpharmaceutical compositions for administration of polypeptides that arewell known in the art and can be adapted readily for administration ofthe present antibodies without undue experimentation.

These antibodies and active fragments thereof, can be used, for example,for specific detection or purification of the novel erbB-3 receptor.Such antibodies could also be used in various methods known in the artfor targeting therapeutic drugs, including cytotoxic agents, to tissueswith high levels of erbB-3 receptors, for example, in the treatment ofappropriate tumors with conjugates of such antibodies and cell killingagents. Accordingly, the present invention further relates to a methodfor targeting a therapeutic drug to cells having high levels of erbB-3receptors, comprising the steps of i) conjugating an antibody specificfor an erbB-3 receptor, or an active fragment of that antibody, to thetherapeutic drug; and ii) administering the resulting conjugate to anindividual with cells having high levels of erbB-3 receptors in aneffective amount and by an effective route such that the antibody isable to bind to the erbB-3 receptors on those cells.

The antibody of this invention is exemplified by rabbit antiseracontaining antibodies which specifically bind to erbB-3 protein. Suchreceptor specific antisera are raised to synthetic peptides representinga unique portion of the erbB-3 amino acid sequence, having six or moreamino acids in sequences which are sufficient to provide a binding sitefor an antibody specific for this portion of the erbB-3 polypeptide.Further, this unique portion of an erbB-3 amino acid sequence, ofcourse, includes sequences not present in an erbB or an erbB-2 aminoacid sequence, as predicted by the respective cDNA sequences. The erbB-3specific anti-peptide antibody of the present invention is exemplifiedby an anti-peptide antibody in polyclonal rabbit antiserum raisedagainst any of the synthetic peptides given in SEQ ID NOS:5-9, which arederived from the predicted sequence of the erbB-3 polypeptide. Thespecific detection of erbB-3 polypeptide with antiserum raised againstthe peptide given in SEQ ID NO:5 is illustrated in mammalian cellstransformed with an expression vector carrying a human erbB-3 cDNA (seeFIG. 7). The antibody of this invention is further exemplified byerbB-3-specific monoclonal antibodies, such as the monoclonal antibodyMAb E3-1, which was raised against the recombinantly expressed proteinand is capable of detecting the native erbB-3 protein. MAb E3-1specifically immunoprecipitated the mature 180 kDa erbB-3 protein fromLTR-erbB-3 transfectants (FIG. 9A) and did not exhibit cross-reactivitywith the EGFR or erbB-2 proteins.

Antibodies to peptides are prepared by chemically synthesizing thepeptides, conjugating them to a carrier protein, and injecting theconjugated peptides into rabbits with complete Freund's adjuvant,according to standard methods of peptide immunization. For example, thepeptide is synthesized by standard methods (Merrifield, R. B., 1963, J.Amer. Soc., 85:2149) on a solid phase synthesizer. The crude peptide ispurified by HPLC and conjugated to the carrier, keyhole limpethemocyanin or bovine thyroglobulin, for example, by coupling the aminoterminal cysteine to the carrier through a maleimido linkage accordingto well-known methods (e.g., Lerner R. A. et al., 1981, Proc. Nat. Acad.Sci. USA, 78:3403). In one standard method of peptide immunology,rabbits are immunized with 100 μg of the erbB-3 peptide-carrierconjugate (1 mg/ml) in an equal volume of complete Freund's adjuvant andthen boosted at 10-14 day intervals with 100 μg of conjugated peptide inincomplete Freund's adjuvant. Additional boosts with similar doses at10-14 day intervals are continued until anti-peptide antibody titer, asdetermined, for example, by routine ELISA assays, reaches a plateau.

The antibody can be labeled with a detectable moiety or attached to asolid support by methods known in the art to facilitate detection of anantibody/antigen complex. Such a detectable moiety will allow visualdetection of a precipitate or a color change, visual detection bymicroscopy, or automated detection by spectrometry or radiometricmeasurement or the like. Examples of detectable moieties includefluorescein and rhodamine (for fluorescence microscopy), horseradishperoxidase (for either light microscopy or electron microscopy andbiochemical detection), biotin-strepavidin (for light or electronmicroscopy) and alkaline phosphatase (for biochemical detection by colorchange). The detection methods and moieties used can be selected, forexample, from the list above or other suitable examples by the standardcritcria applied to such selections (Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1988).

Thus, by following the teachings of the present disclosure, includingapplication of generally known immunological methods cited herein, oneof ordinary skill in the art is able to obtain erbB-3-specificantibodies and use them in a variety of immunological assays, forexample, for diagnostic detection of unusually high or low expression innormal or tumor tissues. Thus, the present invention also relates to abioassay for detecting an erbB-3 antigen in a biological samplecomprising the steps of: i) contacting that sample with an antibody ofthe present invention specific for an erbB-3 polypeptide, underconditions such that a specific complex of that antibody and thatantigen can be formed; and ii) determining the amount of that antibodypresent in the form of those complexes.

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments and the Examplesand Figures included therein.

DESCRIPTION OF THE FIGURES

FIG. 1. Detection of v-erbB-related DNA fragments in DNAs from normalhuman thymus (lane 1), human mammary tumor lines MDA-MB468 (lane 2), andSK-BR-3 (lane 3). Hybridization was conducted at reduced (panel A) orintermediate (panel B) stringency conditions. The arrow denotes a novel9 kilobase pair (kbp) erbB-related restriction fragment distinct fromthose of the EGFR gene (erbB) and erbB-2;

FIG. 2. Genomic and cDNA cloning of erbB-3. The region of (v-erbB)homology within the gcnomic 9 kbp SacI insert of λE3-1 was subclonedinto the plasmid pUC (pE3-1) and subjected to nucleotide sequenceanalysis. The three predicted exons are depicted as solid boxes. erbB-3cDNA clones were isolated from oligo dT-primed libraries of mRNAs fromnormal human placenta (shaded bars) and the breast tumor cell line MCF-7(open bar). The entire nucleotide sequence was determined for bothstrands on erbB-3 complementary DNA from normal human placenta andupstream of the 5′ XhoI site on pE3-16. The coding sequence is shown asa solid bar and splice junctions of the three characterized genomicexons are indicated by vertical white lines. Solid lines in the cDNA maprepresent untranslated sequences. Restriction sites: A=AccI, Av=AvaI,B=BamHI, Bg=BglII, E=EcoRI, H=HindIII, K=KpnI, M=MstII, P=PstI, S=SacI,Sm=SmaI, Sp=SpeI;

FIG. 3. Nucleotide sequence of the region of v-erbB homology in thehuman erbB-3 gene derived from human genomic DNA clone E3-1, in the 1.5kbp region from the EcoRI to the PstI sites. This region contains threeopen reading frames bordered by splice junction consensus sequences(underlined). The predicted amino acid sequences of the three exons areshown in three letter code below the relevant DNA sequences;

FIG. 4. Comparison of the predicted amino acid sequence of the erbB-3polypeptide with other receptor-like tyrosine kinases. The amino acidsequence is shown in single letter code and is numbered on the right.The putative extracellular domain (light shading) extends between thepredicted signal sequence (solid box) at the amino-terminus and a singlehydrophobic transmembrane region (solid box) within the polypeptide. Thetwo cysteine clusters (Cys) in the extracellular domain and thepredicted tyrosine kinase domain (TK) within the cytoplasmic portion ofthe polypeptide are outlined by dark shading. The putative ATP-bindingsite at the amino-terminus of the TK domain is circled. Potentialautophosphorylation sites within the carboxyl-terminal domain (COOH) areindicated by asterisks. Potential N-linked glycosylation sites (→) aremarked above the amino acid sequence. The percentage of amino acidhomology of erbB-3 in individual domains with erbB-2, EGFR, met, eph,insulin receptor (IR), and fins is listed below. Less than 16% identityis denoted by (−);

FIG. 5. Assignment of the genomic locus of erbB-3 was assigned to humanchromosomal locus 12q13. A total of 142 grains were localized on the400-band ideogram. As depicted in the diagram, specific labeling ofchromosome 12 was observed, where 38 out of 51 grains were localized toband q13;

FIG. 6. Elevated erbB-3 transcript levels in human mammary tumor celllines. A Northern blot containing 10 μg total cellular RNA from AB589mammary epithelial cells (lane 1), as well as mammary tumor cell linesMDA-MB415 (lane 2) and MDA-MB453 (lane 3) was hybridized with an erbB-3cDNA probe (panel A). Following signal decay the same blot wasrehybridized with a human β-actin cDNA probe (Gunning, P., Ponte, P.,Okayama, H., Engel, J., Blau, H. & Kedes, L., 1983, Mol. Cell Biol.3:787-795);

FIG. 7. Expression of a human erbB-3 polypeptide in cells transformed bya cDNA segment as detected by an erbB-3-specific anti-peptide antiserum.Cellular lysates (100 μg of each sample) were electrophoresed andtransferred to nitrocellulose membranes for analysis by Westernblotting. Panel A. Detection of erbB-3 polypeptide with the antiserum.Panel B. Preincubation of the antiserum with homologous peptide.Antibody blocking indicates binding specificity. Lane 1: Selectedcultures of NIH3T3 cells transfected with 1 μg LTRerbB-3 expressionvector. Lane 2: control NIH3T3 cells;

FIG. 8. Characterization of gp180^(erbB-3) recombinantly expressed inNIH/3T3 cells. A: Immunoblot analysis of transfectants with erbB-3peptide antisera MK4 and MK5 and peptide competition. B:Immunoprecipitation with MK5 antiserum of LTR-erbB-3 transfectantsmetabolically labeled for 2 h in the presence or absence ofglycosylation inhibitor tunicamycin (1 μg/ml). C: Pulse-chase analysis:LTR-erbB-3 transfectants were pulse labeled for 15 min with 0.5 mCi eachof [³⁵] methionine and [³⁵S] cysteine and immediately lysed (0) orchased with 100 μg/ml each of unlabeled methionine and cysteine for theindicated time periods. 1×10⁷ TCA-precipitable counts wereimmunoprecipitated from total lysates using MK5 antiserum;

FIG. 9. Immunolocalization of gp180^(erbB-3) on the surface ofLTR-erbB-3 cells. A: Immunoprecipitation analysis of metabolicallylabeled LTR-erbB-3 transfectants with monoclonal antibody E3-1 and anon-immune control. B: Indirect immunofluorescence: Formalin-fixedLTR-erbB-3 transfectants were incubated with MAb E3-1 (upper left) ornon-immune IgG (lower left) and stained with a fluorescein-conjugatedsecondary antibody (100× original magnification). Indirectimmunofluorescence with MAb E3-1 of native LTR-erbB-3 cells (rightpanel; 1000× original magnification);

FIG. 10. Autophosphorylation in vitro and chronic tyrosinephosphorylation in vivo of gp180^(erbB-3). LTR-erbB-3 or control lysateswere immunoprecipitated with erbB-3 monoclonal antibody (E3-1) ornon-immune IgG (NI). Parallel immunoprecipitates were subjected eitherto immunoblot analysis with MK4 antiserum (A) or to an immunocomplexkinase assay in the presence of [³²P]-γATP (B). Tyrosine Phosphorylationin vivo was assayed by immunoprecipitation with monoclonal anti-P-Tyrantibodies followed by immunoblotting with MK4 antiserum (C);

FIG. 11. EGF-dependent tyrosine phosphorylation of an EGFR/erbB-3chimeric receptor, gp180^(EGFR/erbB-3). Serum-starved LTR-erbB-3,LTR-EGFR/erbB-3, and LTR-EGFR transfectants were triggered with 100ng/ml EGF. Similar amounts of gp180^(erbB-3), gp180^(EGFR/erbB-3), andEGFR were immunoprecipitated with erbB-3 (E3-1) or EGFR (AB-1)monoclonal antibodies followed by immunoblot analysis with anti-P-Tyrantibodies or peptide antisera; and

FIG. 12. Activation of gp180^(erbB-3) signaling function in human breasttumor cells. The erbB-3 protein was immunoprecipitated with MAb E3-1from 1 mg total protein lysate and subjected to immunoblot analysis witherbB-3 peptide antiserum or phosphotyrosine antibodies as indicated.

DESCRIPTION OF SPECIFIC EMBODIMENTS

As used herein, the terms “polypeptide”, “protein”, “gene product”,“antigen”, “receptor”, “receptor protein” and the like, when used inreference to erbB-3, encompass the erbB-3 amino acid functional sequenceas given in SEQ ID NO:4, the mature erbB-3 glycoprotein, gp180^(erbB-3),and these entities modified by other post-translational modifications,such as glycosylation or tyrosine phosphorylation. However, as is commonin the art, the term “erbB-3 polypeptide” typically refers to thesequence as given in SEQ ID NO:4 while the remaining terms typicallyrefer to gp180^(erbB-3).

The identification of a third member of the erbB-EGF receptor family ofmembrane spanning tyrosine kinases and the cloning of its full lengthcoding sequence is described in the Examples herein. The presence ofapparent structural domains resembling those of the EGF receptorsuggests the existence of an extracellular binding site for a ligand.The structural relatedness of the extracellular domain of the erbB-3receptor with that of the EGF receptor indicates that one or more of anincreasing number of EGF-like ligands (Shoyab, M., Plowman, G. D.,McDonald, V. L. Bradley, J. G. & Todaro, G. J., 1989, Science243:1074-1076) interacts with the erbB-3 product. Accordingly, theerbB-3 gene is expected to play important roles in both normal andneoplastic processes, as is known for the EGFR and erbB-2 genes.

Despite extensive collinear homology with the EGF receptor and erbB-2,distinct regions within the predicted erbB-3 coding sequence revealedrelatively higher degrees of divergence. For example, its carboxylterminal domain failed to exhibit significant collinear identity scoreswith either erbB-2 or EGFR. The divergence at the carboxyl terminus alsoaccounts for minor size differences among the three polypeptides oferbB-3, erbB-2, and EGFR, which possess estimated molecular weights of148 kilodaltons (kd), 138 kd, and 131 kd, respectively. Within thetyrosine kinase domain, which represents the most conserved region ofthe predicted erbB-3 protein, a short stretch of 29 amino acids closerto the carboxyl terminus than the ATP binding site differed from regionsof the predicted erbB-2 and EGFR coding sequence in 28 and 25 positions,respectively. Such regions of higher divergence in their cytoplasmicdomains are likely to confer different functional specificity to theseclosely related receptor-like molecules. Thus, mutations or otheralterations in expression of the erbB-3 gene are likely to cause cancersor genetic disorders different from those associated with such defectsin the erbB and erbB-2 genes.

Chromosomal mapping localized erbB-3 to human chromosome 12 at theq11-13 locus, whereas the related EGFR and erbB-2 genes are located atchromosomal sites 7p12-13 and 17p12-21.3, respectively. Thus, each geneappears to be localized to a region containing a different homeobox anda different collagen chain gene locus. Keratin type I and type II genesalso map to regions of 12 and 17, respectively, consistent with thedifferent localizations of erbB-3 and erbB-2, respectively. Thus, theDNA segments of the present invention represent novel probes to aid ingenetic mapping of any heritable diseases which are associated withchromosomal aberrations in the vicinity of the 12q11-13 locus.

There is evidence for autocrine as well as paracrine effectors of normalcell proliferation. The former are factors that are produced by the samecells upon which they stimulate cell proliferation, whereas the latterfactors are secreted by cells other than those that are affected bythose factors. However, the inherent transforming potential of autocrinegrowth factors suggests that growth factors most commonly act on theirtarget cell populations by a paracrine route. The present survey oferbB-3 gene expression indicates its normal expression in cells ofepithelial and neuroectodermal derivation. Comparative analysis of thethree erbB receptor-like genes in different cell types of epidermaltissue revealed that keratinocytes expressed all three genes. Incontrast, melanocytes and stromal fibroblasts specifically lacked EGFRand erbB-3 transcripts, respectively. Thus, melanocytes and stromalfibroblasts may be sources of paracrine growth factors for EGFR anderbB-3 products, respectively, that are expressed by the other celltypes residing in close proximity in epidermal tissues.

Given that both erbB and erbB-2 have been causally implicated in humanmalignancy, the present findings (Example 5) that the erbB-3 transcriptis overexpressed in a significant fraction of human mammary tumor celllines indicates that this new member of the EGFR receptor family alsoplays an important role in some human malignancies.

Characterization of the human erbB-3 gene product, gp180^(erbB-3), showsthat it is a transmembrane glycoprotein exhibiting propertiescharacteristic of a receptor-like tyrosine kinase. The recombinant humanerbB-3 protein shared identical electrophoretic mobility with thenatural erbB-3 product expressed in human breast tumor cell lines.Moreover, both recombinant and endogenously expressed gp180^(erbB-3)were recognized by different antibodies directed against distinctepitopes, such as monoclonal (i.e., Mab E3-1) and peptide antibodiesdirected against epitopes in the extracellular and carboxyl-terminaldomains. The 145 kDa erbB-3 polypeptide precursor conformed withpredicted erbB-3 encoded protein following cleavage of its signalsequence. Finally, demonstration of its inherent signaling propertiesestablished functional integrity of recombinantly expressedgp180^(erbB-3).

Although the erbB-3 tyrosine kinase domain shares greater than 60% aminoacid identity with the EGFR and erbB-2 proteins, single amino acidsubstitutions differences in highly conserved residues shared amongknown tyrosine kinases raised a question as to whether erbB-3 harborsintrinsic catalytic activity involved in signal propagation instead ofsignal attenuation as has been postulated for certain receptor tyrosinekinase-like molecules (Chou et al., Proc. Natl. Acad. Sci. USA 88:4897(1991)). Most notably, codon 834 within the tyrosine kinase domainpredicts asparagine in erbB-3, while aspartate is present at thisposition in essentially all known protein kinases. Moreover,substitution of asparagine for aspartate in this position abolishesc-kit and v-fps tyrosine kinase activity. The present characterizationof the erbB-3 cytoplasmic domain demonstrates not only its catalyticfunction but also the ability to transduce a mitogenic signal as well.gp180^(erbB-3) demonstrated autokinase activity in vitro and, in somecell lines, tyrosine phosphorylation in vivo. Moreover, EGF-dependentactivation of gp180^(EGFR/erbB-3) was associated both with mitogenicsignaling and in vivo tyrosine phosphorylation of the chimeric receptor.All of these findings imply that the erbB-3 protein represents abiologically active membrane spanning receptor capable of transducing amitogenic signal in a ligand-dependent manner. Thus, the erbB-3 geneencodes a membrane spanning molecule possessing all the properties of afunctional growth receptor.

Constitutive activation of erbB-3 catalytic activity was demonstrated inLTR-erbB-3 transfectants. These results raise the possibility thatNIH/3T3 cells may express an erbB-3 ligand. If so, this putative ligandwould unlikely interact with the EGFR, since overexpression of thelatter in NIH/3T3 cells is not associated with its chronic tyrosinephosphorylation in the absence of exogenous EGF. This invention furtherestablished that EGF neither enhanced in vivo tyrosine phosphorylationof gp180^(erbB-3) nor elicited a mitogenic response in LTR-erbB-3 cells.Additional ligands of the EGF family, including TGFα, amphiregulin, andHB-EGF, have also failed to stimulate gp180^(erbB-3) tyrosinephosphorylation or DNA synthesis in LTR-erbB-3 cells. While a lowaffinity interaction of known EGF-related ligands for gp180^(erbB-3)cannot be excluded, these findings indicated that erbB-3 and EGFRproteins possess distinct ligand specificities. The ability to triggerthe erbB-3 catalytic domain in the EGFR/erbB-3 chimeric molecule shouldmake it possible to more readily identify its substrates as well as tocompare them with those of its closely related family members.

Based upon this invention's demonstration that the erbB-3 protein isboth catalytically active and can elicit a proliferative response inNIH/3T3 cells, the instant findings of its chronic activation in somehuman breast tumor cells suggest its contribution to the malignantphenotype in such tumors.

Analogous evidence has implicated overexpression associated with geneamplification of both EGFR and erbB-2 in a variety of tumors as well. Insuch tumors, there is precedence for activation of receptor kinaseactivity by mechanisms involving autocrine loops as well as geneticalterations affecting regulatory or coding sequences.

Both EGFR and erbB-2 genes have been implicated as oncogenes based upondemonstration of their overexpression and constitutive activation invarious human tumors. The results of this invention argue strongly thatthe most recently identified family member, erbB-3, is activated in somehuman breast tumors. Overexpression of the erbB-3 protein did notinvariably correlate with its chronic tyrosine phosphorylation. Hence,erbB-3 activation may involve autocrine stimulation or subtle geneticalterations. In addition to breast tumors, expression of the erbB-3transcript has been observed in a wide range of human carcinomas,including colon, lung, kidney, pancreas, and skin. These findings promptthe search for evidence of erbB-3 activation as an oncogene in theseother common human cancers.

EXAMPLE 1 Identification of a Human DNA Fragment Related to the erbB-3Proto-oncogene Family

In an effort to detect novel erbB-related genes, human genomic DNA wascleaved with a variety of restriction endonucleases and subjected toSouthern blot analysis with v-erbB as a probe. Normal mammary epithelialcells AB589 (Walen, K. H. & Stampfer, M. R., 1989, Cancer. Genet.Cytogenet. 37:249-261) and immortalized keratinocytes RHEK have beendescribed previously (Rhim, J. S., Jay, G., Arnstein, P., Price, F. M.,Sanford, K. K. & Aaronson, S. A., 1985, Science 227:1250-52). Normalhuman epidermal melanocytes (NHEM) and keratinocytes (NHEK) wereobtained from Clonetics. Sources for human embryo fibroblasts (Rubin, J.S., Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S., & Aaronson, S.A., 1989, Proc. Nat. Acad Sci. USA 86:802-806) or mammary tumor celllines SK-BR-3, MDA-MB468, MDA-MB453, and MDA-MB415 (Kraus, M. H.,Popescu, N. C., Amsbaugh, S. C. & King, C. R., 1987, EMBO. J 6:605-610)have been described. For nucleic acid RNA hybridization, DNA and RNAwere transferred to nitrocellulose membranes as previously described(Kraus, M. H., et al., 1987, supra). High stringency hybridization wasconducted in 50% formamide and 5×SSC at 42° C. Filters were washed at50° C. in 0.1×SSC. Reduced stringency hybridization of DNA was carriedout in 30% formamide followed by washes in 0.6×SSC, while intermediatestringency was achieved by hybridization in 40% formamide and washing in0.25×SSC. For the specific results depicted in FIG. 1, DNAs wererestricted with SacI and hybridized with probe specific for an oncogenicviral form of the erbB gene, v-erbB, spanning from the upstream BamHIsite to the EcoRI site in the avian erythroblastosis proviral DNA(Vennstrom, B., Fanshier, L., Moscovici, C. & Bishop, J. M., 1980, J.Virol. 36:575-585).

Under reduced stringency hybridization, four SacI restriction fragmentswere detected. Two were identified as EGFR gene fragments by theiramplification in the mammary tumor cell line MDA-MB468 (FIG. 1A, lane1,2) known to contain EGFR gene amplification and one as an erbB-2specific gene fragment due to its increased signal intensity in anothermammary tumor cell line, SK-BR-3, known to have erbB-2 amplified (FIG.1A, lane 1,3). However, a single 9 kbp SacI fragment exhibited equalsignal intensities in DNAs from normal human thymus, SK-BR-3 and a linewith high levels of EGFR, A431 (FIG. 1A). When the hybridizationstringency was raised by 7° C., this fragment did not hybridize, whereasEGFR and erbB-2 specific restriction fragments were still detected withv-erbB as a probe (FIG. 1B). Taken together, these findings suggestedthe specific detection of a novel v-erbB-related DNA sequence within the9 kbp SaI fragment.

EXAMPLE 2 Cloning of the Human DNA Fragment Related to erbB

For further characterization, a normal human genomic library wasprepared from SacI cleaved thymus DNA enriched for 8 to 12 kbpfragments. For convenience, bacteriophage λsep-lacS was obtained from L.Prestidge and D. Hogness (Stanford University); many other cloningvectors derived from phage λ or other genomes can be used for cloningthis DNA fragment according to standard recombinant DNA methods that arewell known in the art. Purified phage DNA was subjected to cos-endligation, restriction with SacI, and fractionation in a continuous10-40% sucrose gradient. A genomic library was prepared by ligating SacIrestriction fragments of normal human thymus DNA in the molecular weightrange of 8 kbp to 12 kbp (isolated by sucrose gradient sedimentation)with the purified phage arms. Ten recombinant clones detected by v-erbBunder reduced stringency conditions did not hybridize with human EGFR orerbB-2 cDNA probes at high stringency. As shown in the restriction mapof a representative clone with 9 kbp insert, the region of v-erbBhomology was localized by hybridization analysis to a 1.5 kbp segmentspanning from the EcoRI to the downstream PstI site.

The nucleotide sequence of a portion of a clone of the novel humangenomic DNA fragment related to erbB was determined for both DNA strandsby the dideoxy chain termination method (Sanger, F., Nicklen, S. &Coulson, A. R., 1977, Proc. Nat. Acad. Sci. USA. 74:5463-67) usingsupercoiled plasmid DNA as template. The nucleotide sequence wasassembled and translated using IntelliGenetics software. Amino acidsequence comparison was performed with the alignment program by Pearsonand Lipman (Pearson, W. R. & Lipman, D. J., 1988, supra) as implementedon the computers of the NCI Advanced Scientific Computing Laboratory.Hydrophobic and hydrophilic regions in the predicted protein wereidentified according to Kyte and Doolittle (Kyte, J. & Doolittle, R. F.,1982, J. Mot Biol. 157:105-132). Nucleotide sequence analysis revealedthat the region of v-erbB homology in the 1.5 kbp segment from the EcorIto the PstI contained three open reading frames bordered by splicejunction consensus sequences (FIG. 2). Computerized comparisons of thepredicted amino acid sequence of these three open reading frames withother known proteins revealed the highest identity scores of 64% to 67%to three regions which are contiguous in the tyrosine kinase domains ofv-erbB, as well as human EGFR and erbB-2 proteins. Furthermore, allsplice junctions of the three characterized exons in the new gene wereconserved with erbB-2. Amino acid sequence homology to other knowntyrosine kinases was significantly lower, ranging from 39% to 46%.

A single 6.2 kb specific mRNA was identified by Northern blot analysisof human epithelial cells using the 150 bp Spel-AccI exon-containingfragment as probe (FIG. 2). Under the stringent hybridization conditionsemployed, this probe detected neither the 5 kb erbB-2 mRNA nor the 6 kband 10 kb EGFR mRNAs. All of these findings suggested that the presentwork has identified a new functional member of the erbB proto-oncogenefamily, which tentatively has been designated as erbB-3.

EXAMPLE 3 Cloning and Characterization of cDNAs for the mRNA of theHuman erbB-3 Gene

In an effort to characterize the entire erbB-3 coding sequence,overlapping cDNA clones were isolated from oligo dT-primed cDNAlibraries from sources with known erbB-3 expression, utilizinggene-specific genomic exons or cDNA fragments as probes. In brief, anoligo dT-primed human placenta cDNA library in λgt11 was obtained fromClontcch. MCF-7 cDNA was prepared by first strand synthesis from 5 μgpoly A⁺ RNA using an oligo dT containing linker-primer and Mo-MuLVreverse transcriptase, followed by second strand synthesis with DNApolymerase I, RNaseH, and subsequent T4 DNA polymerase treatment.Double-stranded cDNA was directionally cloned into the SfiI site ofλpCEV9 using specific linker-adapter oligonucleotides (Miki, T., Matusi,T., Heidaran, M. A. & Aaronson, S. A., 1989, Gene 83:137-146; see also,U.S. application Ser. No. 07/386,053 of Miki et al., filed Jul. 28,1989). Following plaque purification, phage DNA inserts were subclonedinto pUC-based plasmid vectors for further characterization. The cloneswere initially characterized by restriction analysis and hybridizationto the mRNA, and were subsequently subjected to nucleotide sequenceanalysis. Clones designated pE3-6, pE3-8, pE-9, and pE3-11 carryinginserts with molecular weights ranging from 13 kbp to 43 kbp wereisolated from a human placenta library, whereas the pE3-16 clonecontaining a 5 kbp insert was obtained by screening the MCF-7 cDNAlibrary with the upstream most coding sequence of pE3-11 as a probe. Theclones pE3-8, pE3-9, pE3-11, and pE3-16 contained identical 3′ endsterminating in a poly A stretch (FIG. 2).

The complete coding sequence of erbB-3 was contained within a singlelong open reading frame of 4080 nucleotides extending from position 46to an in-frame termination codon at position 4126. The most upstream ATGcodon at position 100 was the likely initiation codon, as it waspreceded by an in-frame stop codon at nucleotide position 43 andfulfilled the criteria of Kozak for an authentic initiation codon. Theopen reading frame comprised 1342 codons predicting a 148 kdpolypeptide. Downstream from the termination codon, multiple stop codonswere present in all frames. As shown in SEQ ID NO:4, the deduced aminoacid sequence of the erbB-3 polypeptide predicted a transmembranereceptor tyrosine kinase most closely related to EGFR and erbB-2. Ahydrophobic signal sequence of erbB-3 was predicted to comprise the 19amino-terminal amino acid residues. Cleavage of this signal sequencebetween glycine at position 19 and serine at position 20 would generatea processed polypeptide of 1323 amino acids with an estimated molecularweight of 145 kd. A single hydrophobic membrane spanning domainencompassing 21 amino acids was identified within the coding sequenceseparating an extracellular domain of 624 amino acids from a cytoplasmicdomain comprising 678 amino acids (SEQ ID NO:4).

The putative erbB-3 ligand-binding domain was 43% and 45% identical inamino acid residues with the predicted erbB-2 and EGFR protein,respectively. Within the extracellular domain, all 50 cysteine residuesof the processed erbB-3 polypeptide were conserved and similarly spacedwhen compared to the EGFR and erbB-2. Forty-seven cysteine residues wereorganized in two clusters containing 22 and 25 cysteines respectively, astructural hallmark of this tyrosine kinase receptor subfamily (see, forexample, Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N.,Miyajima, N., Saito, T. and Toyoshima, K, 1986, Nature 319:230-234). Tenpotential N-linked glycosylation sites were localized within the erbB-3extracellular domain. In comparison with the EGFR and erbB-2 proteins,five and two of these glycosylation sites were conserved, respectively.Among these, the site proximal to the transmembrane domain was conservedamong all three proteins (SEQ ID NO:4).

Within the cytoplasmic domain, a core of 277 amino acids from position702 through 978 revealed the most extensive homology with the tyrosinekinase domains of EGFR and erbB-2. In this region 60% or 62% of aminoacid residues were identical and 90% or 89% were conserved,respectively. This stretch of amino acid homology coincides with theminimal catalytic domain of tyrosine kinases (Hanks, S. K., Quinn, A. M.& Hunter, T., 1988, Science 241:42-52).

There was significantly lower homology with other tyrosine kinases (FIG.4). The consensus sequence for an ATP-binding site (GxGxxG, Hanks, S. K.et al., 1988, supra) was identified at amino acid positions 716 through721. This sequence as well as a lysine residue located 21 amino acidresidues further toward the carboxyl terminus was conserved between thethree erbB-related receptors. Taken together these findings defined theregion between amino acid position 702 and 978 as the putative catalyticdomain of the erbB-3 protein (SEQ ID NO:4).

The most divergent region of erbB-3 compared to either EGFR or erbB-2was its carboxyl terminus comprising 364 amino acids. This region showeda high degree of hydrophilicity and the frequent occurrence of prolineand tyrosine residues. Among these tyrosine residues, those at positions1197, 1199, and 1262 matched closest with the consensus sequence forputative phosphorylation sites. The peptide sequence YEYMN (SEQ IDNO:12), encompassing tyrosine 1197 and 1199, was repeated at positions1260-1264 and was at both locations surrounded by charged residues,providing an environment of high local hydrophilicity. Theseobservations render tyrosines 1197, 1199 and 1262 likely candidates forautophosphorylation sites of the erbB-3 protein.

EXAMPLE 4 Chromosomal Mapping of the Human erbB-3 Gene

The chromosomal location of the erbB-3 gene was determined by in situhybridization (Popescu, N. C., King, C. R. & Kraus, M. H., 1989,Genomics 4:362-366) with a ³H-labeled plasmid containing theamino-terminal erbB-3 coding sequence. A total of 110 human chromosomespreads was examined prior and subsequent to G banding foridentification of individual chromosomes. A total of 142 grains waslocalized on a 400-band ideogram. Specific labeling of chromosome 12 wasobserved, where 38 out of 51 grains were localized to band q13 (FIG. 5).Thus, the genomic locus of erbB-3 was assigned to 12q13. In this regionof chromosome 12, several genes have previously been mapped includingthe melanoma-associated antigen ME491, histone genes and the gene forlactalbumin. In addition, two proto-oncogenes, int-1 and gli are locatedin close proximity to erbB-3.

EXAMPLE 5 erbB-3 Expression in Normal and Malignant Human Cells

To investigate its pattern of expression, a number of human tissues weresurveyed for the erbB-3 transcript. The 6.2 kb erbB-3 specific mRNA wasobserved in term placenta, postnatal skin, stomach, lung, kidney, andbrain, while it was not detectable in skin fibroblasts, skeletal muscleor lymphoid cells. Among the fetal tissues analyzed, the erbB-3transcript was expressed in liver, kidney, and brain, but not in fetalheart or embryonic lung fibroblasts. These observations indicate thepreferential expression of erbB-3 in epithelial tissues and brain.

ErbB-3 expression was also investigated in individual cell populationsderived from normal human epithelial tissues including keratinocytes,glandular epithelial cells, melanocytes, and fibroblasts. For comparisonlevels of EGFR and erbB-2 transcripts were analyzed. As shown in Table1, erbB-3 mRNA levels were relatively high in keratinocytes, comparablewith those of erbB-2 and EGFR in these cells. Lower, but similarexpression levels of each transcript were detected in cells derived fromglandular epithelium. These findings are consistent with growthregulatory roles of all three receptor-like molecules in squamous andglandular epithelium. Whereas erbB-2 and EGFR transcripts were alsoreadily observed in normal fibroblasts, the same cells lacked detectableerbB-3 mRNA. In contrast, normal human melanocytes, which expressed botherbB-3 and erbB-2 at levels comparable with human keratinocytes, lackeddetectable EGFR transcripts. Thus, the expression patterns of thesereceptor-like molecules were different in specialized cell populationsderived from epidermal tissues.

TABLE 1 Normal expression pattern of human erbB gene family members CellSource of Transcripts Gene Relative RNA levels Embryonic fibroblast(M426) erbB-3 − erbB-2 + EGF-R + Skin fibroblast (501T) erbB-3 −erbB-2 + EGF-R + Immortal keratinocyte (RHEK) erbB-3 ++ erbB-2 ++ EGF-R++ Primary keratinocyte (NHEK) erbB-3 + erbB-2 + EGF-R ++ Glandularepithelium (AB589) erbB-3 (+) erbB-2 (+) EGF-R (+) Melanocyte (NHEM)erbB-3 ++ erbB-2 ++ EGF-R − Replicate Northern blots were hybridizedwith equal amounts (in cpm) of probes of similar specific activities forerbB-3, erbB-2, and EGFR, respectively. Relative signal intensities wereestimated: − not detectable, (+) weakly positive, + positive, ++strongly positive.

To search for evidence of erbB-3 involvement in the neoplastic process,erbB-3 mRNA levels in a series of human tumor cell lines were surveyed.The erbB-3 transcript was detected in 36 of 38 carcinomas of 2 of 12sarcomas while 7 tumor cell lines of hematopoietic origin lackedmeasurable erbB-3 mRNA. Markedly elevated levels of a normal-sizedtranscript were observed in 6 out of 17 tumor cell lines derived fromhuman mammary carcinomas. By Southern blot analysis, neither gross generearrangement nor amplification was detected in the cell lines. FIG. 6Ashows the results of Northern blot analysis with control AB589nonmalignant human mammary epithelial cells (lane 1) and tworepresentative human mammary tumor lines, MDA-MB415 (lane 2) andMDA-MB453 (lane 3). Hybridization of the same filter with human β-actinprobe (FIG. 6B) verified actual levels of mRNA in each lane.Densitometric scanning indicated that the erbB-3 transcript in eachtumor cell line was elevated more than 100 fold above that of thecontrol cell line. Thus, overexpression of this new member of the erbBfamily, as in the case of the EGFR and erbB-2 genes, is likely to playan important role in some human malignancies.

EXAMPLE 6 Further Characterization of the normal erbB-3 Gene Product

The pZIPneo expression vector (Cepko et al, Cell 37:1053 (1984)) wasmodified by introduction of a unique Sal I cloning site. Followingdeletion of the Sal I site in the tetracyline resistance gene, thesynthetic oligonucleotides 5′-GATCTCGAGTCGAC-3′ (SEQ ID NO:10) and5′-GATCGTCGACTCGA-3′ (SEQ ID NO:11) were annealed and ligated into thesingle Bam HI site to generate pZIPneo_(Sal). The erbB-3 open readingframe including 7 nucleotides upstream of the initiation codon and thetermination codon (nucleotides 93-4128) was linkered with Sal I ends,employing the polymerase chain reaction (PCR) and cloned intopZIPneo_(Sal)(LTR-erbB). Sense orientation and integrity of the openreading frame were confirmed by restriction analysis as well asnucleotide sequence analysis of cloning boundaries and PCR-amplifiedregions.

For structural and functional characterization of the erbB-3 geneproduct, the complete erbB-3 open reading-frame was inserted as givenabove into the modified ZIPnco vector, placing the cDNA under thetranscriptional control of the Moloney murine leukemia viruslong-terminal-repeat sequence (LTR-erbB-3). NIH/3T3 fibroblasts weretransfected with LTR-erbB-3 or LTR-neo control DNA and cultured in thepresence or absence of the selective drug G418. Under conditions inwhich efficient drug resistance (6×10³ colonies/pmol) was conferred byLTR-erbB-3, no transformed foci were detectable. In contrast, LTR-erbB-2or EGF-triggered LTR-EGFR induced morphological transformation ofNIH-3T3 cells with efficiencies of around 1.2×10⁴/pmol and 2.3×10²/pmol,respectively.

To test for expression of the erbB-3 protein, polyclonal rabbitantisera, including MK4 and MK5, were developed against syntheticpeptides. MK4 and MKS were raised against peptides that encompass theresidues given in SEQ ID NO:5 and SEQ ID NO:6, respectively, which arewithin the carboxyl terminus of the predicted erbB-3 product. Forimmunization, peptides were coupled to thyroglobulin usingglutaraldehyde. Immunoblot analysis of lysates from marker-selectedLTR-neo and LTR-erbB-3 transfectants revealed a major 180 kDa band onlyin LTR-erbB-3 cells. This band was independently recognized by bothantisera (FIG. 8A). There was no cross-reactivity of either antiserumwith the related EGFR or erbB-2 proteins overexpressed in NIH/3T3 cells.Immunoreactivity of either antiserum with the 180 kDa band in LTR-erbB-3transfectants was competed by the antigenic peptide, while MK4reactivity with a faint 125 kDa band was not affected by preincubationwith peptide (FIG. 8A). These results established specificity of erbB-3protein detection by both polyclonal antisera. By comparison, the 180kDa erbB-3 protein migrated distinctly slower than the 170 kDa EGFR andslightly faster than the 185 kDa erbB-2 protein.

To characterize processing of the erbB-3 protein, we performedimmunoprecipitation experiments with MK5 antiserum. Metabolic labelingof LTR-erbB-3 transfectants in the presence or absence of tunicamycindemonstrated that the 145 kDa erbB-3 core polypeptide is modified byN-linked glycosylation (FIG. 8B). Pulse-chase analysis further indicatedcotranslational processing, resulting in a predominant 170 kDa precursorprotein in addition to faint erbB-3 specific bands of 150 kDa and 160kDa, following 15 min of pulse-labeling (FIG. 8C). The mature 180 kDaerbB-3 protein appeared after 0.5 h of chase, and the majority wasconverted into gp180^(erbB-3) by 1 h. By analysis of further timepoints, we estimate an approximate half-life of 2-3 h (FIG. 8C). Thus,in NIH/3T3 cells, gp180^(erbB-3) exhibits an apparently faster turn-overthan the EGFR, for which a biosynthesis time of 3h and approximatehalf-life of 3-6 h has been reported.

For immunolocalization of the erbB-3 protein, erbB-3-specific monoclonalantibodies, including MAb E3-1, were raised against the recombinantlyexpressed protein. BALB/c mice were immunized with live LTR-erbB-3cells. Somatic cell hybrids were prepared by fusion of immunesplenocytes with murine non-secreting myeloma cells NS-1. Hybridomasupernatants were screened for differential immunoreactivity withLTR-erbB-3 but not LTR-neo transfectants by enzyme-linked immunosorbentassay (ELISA) using both live cells or cell extracts as antigen source.Positive hybridoma cell lines were cloned twice by limiting dilution andfurther characterized by immunoprecipitation and immunofluorescenceanalysis. One monoclonal antibody, MAb E3-1 (IgG2a isotype),specifically immunoprecipitated gp180^(erbB-3) from LTR-erbB-3transfectants (FIG. 9A) and did not exhibit cross-reactivity with theEGFR or erbB-2 proteins overexpressed in an NIH/3T3 cell background.Immunofluorescence analysis using a labeled second antibody revealedheterogeneous membrane immunostaining of formalin-fixed LTR-erbB-3 cellsusing MAb E3-1, but not with a non-specific immunoglobulin of matchingisotype (FIG. 9B). MAb E3-1-specific membrane fluorescence of nativeLTR-erbB-3 cells (FIG. 9B) indicated that gp180^(erbB-3) was expressedat the cell surface, as expected for a membrane-anchored protein.

To investigate its function, we next analyzed the erbB-3 protein for invitro kinase activity. LTR-erbB-3 and control LTR-neo cell lysates werefirst immunoprecipitated with E3-1 followed by immunoblot analysis withMK4 antiserum (FIG. 10A). When the same immunoprecipitates wereincubated in autokinase buffer containing [³²P]-γATP, a predominant 180kDa phosphoprotein was labeled only in immunoprecipitates containing theerbB-3 protein (FIG. 10B). These findings indicated that gp180^(erbB-3)possessed intrinsic protein kinase activity. To assess its enzymaticactivity in vivo, LTR-erbB-3 lysates were subjected toimmunoprecipitation with phosphotyrosine-specific monoclonal antibodies(anti-P-Tyr) followed by immunoblotting with MK4 antiserum. As shown inFIG. 10C, the erbB-3 protein was recovered from anti-P-Tyrimmunoprecipitates, and immunodetection was competed either by phenylphosphate in the immunoprecipitation or the erbB-3 peptide in Westernblot analysis. These findings indicated that recombinant gp180^(erbB-3)expressed in NIH/3T3 cells was chronically phosphorylated on tyrosineresidues.

The protein lysates were prepared in Staph A buffer containing theprotease inhibitors phenylmethyl sulfonyl fluoride (1 mM) and aprotinin(10 μg/ml; Boehringer Mannheim). For the analysis of phospho-tyrosineproteins, the phosphatase inhibitors sodium orthovanadate (2 mM) andsodium pyrophosphate (10 mM) were added. Immunoblot analysis usingpeptide antisera was essentially conducted as previously reported. Forthe detection of phosphotyrosine proteins, membranes were blocked in PBScontaining 5% BSA and immunostained with a mixture of monoclonalanti-P-Tyr antibodies (PY20 and PY69; ICN) diluted 1:500 in PBScontaining 1% BSA Filters were washed with PBS containing 0.05 %Tween20. Immunoprecipitation was conducted using gammabind G agarose(Pharmacia) to collect the immunocomplexes. The beads were coupled withgoat anti-mouse-IgG second antibody (Boehringer Mannheim) inimmunoprecipitations using erbB-3 or EGFR monoclonal antibodies. For invitro kinase assays, 4 mg total lysates were precleared with gammabind Gagarose. Following immunoprecipitation, washed immunocomplexes wereequilibrated in autokinase buffer containing 40 mM Hepes 7.5, 10 mMMgCl₂, and 0.05 % Triton. The immunocomplexes were subsequently dividedfor immunoblot analysis or immunocomplex kinase assay, respectively.Autokinase reactions were carried out in 40 μl autokinase buffercontaining 20 Ci γATP (3000 ci/mmol) at 25° for 10 min and terminated byaddition of SDS containing sample buffer.

EXAMPLE 7 EGF-dependent Mitogenic Signaling by an EGFR/erb B-3 ChimericReceptor

To explore erbB-3 signaling, a chimeric receptor, LTR-EGFR/erbB-3,containing the ligand-binding domain of the closely related EGF receptor(aa 1-682) and the intracellular portion of erbB-3 (aa 681-1342) wasengineered. Linearized expression constructs (0.01-10 μg/plate) weretransfected into NIH/3T3 cells by calcium phosphate precipitation using40 μg of calf thymus DNA as carrier. Mass cultures expressing therecombinant proteins were obtained by selection with 750 μg/ml G418.Selected LTR-EGFR/erbB-3 transfectants were enriched for expression ofthe chimeric protein by preparative FACS sorting using EGFR monoclonalantibody AB-1 (Oncogene Sciences).

Transfection of NIH/3T3 cells with this construct did not result indetectable focus formation either in the presence or absence of EGF. Toquantitate expression of the chimeric receptor, selected mass cultureswere analyzed for EGF-binding in comparison to NIH/3T3 cellsoverexpressing the EGFR (LTR-EGFR). Scatchard analysis establishedaround 5.7×10⁵ EGF binding sites/cell for the LTR-EGFR/erbB-3transfectant as compared to 2.5×10⁶ binding sites/cell for LTR-EGFRtransfectant. The LTR-EGFR/erbB-3 transfectant exhibited two populationsof binding sites with affinities of 0.11 nM and 5 nM, respectively. Thehigh-affinity sites were in the minority (2.3×10⁴), and there were5.5×10⁵ low-affinity binding sites. Similar results were obtained withthe wild-type EGFR in LTR-EGFR transfectants, which displayed 1.1×10⁵high affinity (0.13 nM) and 2.4×10⁶ low affinity receptors (7 nM).

To investigate EGF responsiveness of erbB-3 enzymatic activity, the invivo tyrosine phosphorylation of the chimeric receptor in the presenceor absence of EGF was compared. This protein, as well as the EGFR anderbB-3 proteins from independent transfectants were enriched byimmunoprecipitation and subjected to immunoblot analysis with eitheranti-P-Tyr or the appropriate specific antiserum. As shown in FIG. 11,the steady state level of tyrosine phosphorylated gp180^(erbB-3) inNIH/3T3 cells was not altered upon EGF exposure (lane 1,2). The chimericEGFR/erbB-3 receptor, which was expressed as a 180 kDa protein, gp₁₈₀^(EGFR/erbB-3) displayed low, but detectable level of tyrosinephosphorylation in serum-free medium (lane 3). However, EGF triggeringof the chimera resulted in a substantial increase in tyrosinephosphorylation, demonstrating EGF-dependent activation of erbB-3catalytic function (lane 4). The wild-type EGFR showed somewhat higherlevel of EGF-dependent tyrosine phosphorylation under the sameconditions (lane 6). Of note, the relative level of gp180^(erbB-3)tyrosine phosphorylation was comparable to that of EGF-activatedchimeric receptor expressed at a similar protein level, indicatingconstitutive activation of erbB-3 catalytic properties in LTR-erbB-3transfectants.

Whether the erbB-3 catalytic domain was capable of transducing amitogenic signal was then assessed. When the LTR-EGFR/erbB-3transfectant was exposed to increasing EGF concentrations, there was adose-dependent stimulation of DNA synthesis similar to that observedwith EGFR overexpressing NIH-3T3 cells. Under the same conditions,neither LTR-neo nor LTR-erbB-3 transfectants showed a significantincrease in DNA synthesis even at high EGF concentrations, consistentwith previous observations. It should be noted that basal levels of DNAsynthesis of the LTR-erbB-3 transfectant were 2-3 fold above those ofthe other transfectants, findings that were reproducible with severalindependent selected mass cultures.

The biological effects of activated erbB-3 catalytic function wereassessed by testing the transfectants for anchorage-independent growth.To test anchorage-independent growth, cell suspensions were seeded at10-fold serial dilutions in semisolid agarose medium containing growthmedium and 0.45% seaplaque agarose (FMC Corp.). Visible coloniescomprising >100 cells were scored at 14 days. EGF was added at aconcentration of 20 ng/ml. Human mammary tumor cell lines were obtainedfrom the American Type Culture Collection and propagated in Dulbecco'smodified Eagle medium containing 10% fetal calf serum.

As shown in Table 2, LTR-neo transfectants failed to exhibit significantsoft agar growth in the presence or absence of EGF. In contrast, EGFinduced soft agar colony formation with both LTR-EGFR/erbB-3 andLTR-EGFR transfectants. The latter showed a larger colony number (Table2) as well as colony size (data not shown). By comparison, theLTR-erbB-3 transfectant displayed EGF-independent colony formation withan efficiency similar to that of EGF-activated LTR-EGFR/erbB-3transfectant (Table 2). All of these findings establish that ligandactivation of a chimeric EGFR/erbB-3 receptor causes mitogenic signalingin NIH/3T3 cells and suggest that chronic tyrosine phosphorylation oferbB-3 in LTR-erbB-3 transfectants is associated with constitutivesignaling in these cells.

TABLE 2 Anchorage-independent growth of NIH/3T3 transfectants#colonies*/10⁴ cells NIH3T3 transfectants − EGF + EGF LTR-neo 1 (±1) 4(±2) LTR-EGFR 2 (±2) 206 (±49) LTR-EGFR/erbB-3 7 (±3) 88 (±16)LTR-erbB-3 97 (±20) 94 (±29) *mean (± standard error) of 3 independentassays

EXAMPLE 8 Evidence for Activated erbB-3 Signaling Function in HumanBreast Tumor Cells

The availability of erbB-3 specific antibodies made it possible toexplore expression and activity of gp180^(erbB-3) in human tumor cells.Based upon our previous evidence for erbB-3 mRNA overexpression incertain breast cancer cell lines, we measured erbB-3 protein levels andtyrosine phosphorylation in such tumor lines, using the procedures givenabove. Following immunoprecipitation with Mab E3-1, immunoblot analysiswith MK4 antiserum revealed the natural human erbB-3 product as a 180kDa protein. The levels of erbB-3 protein varied markedly along thetumor lines analyzed, with highest expression in BT483, MDA-MB453,MDA-MB134, MDA-MB361, SK-BR-3, and MDA-MB468 (FIG. 12). The lowestlevels were observed in BT20 and MDA-MB175 cell lines (FIG. 12),comparable with that expressed by nonmalignant 184B5 mammary epithelialcells (data not shown). Thus, erbB-3 protein expression varied by atleast 20-30 fold among the lines tested, consistent with results oftranscript analysis (data not shown).

Immunoblot analysis of the same immunoprecipitates with anti-P-Tyrantibodies revealed that tyrosine phosphorylation of the native erbB-3product was undetectable in 8 of the tumor cell lines, includingMDA-MB134, MDA-MB361, and MDA-MB468, which harbored increased erbB-3levels. In contrast, erbB-3 protein expressed by 4 cell lines, includingMDA-MB453, BT474, MDA-MB175, and SK-BR-3, demonstrated readilydetectable chronic tyrosine phosphorylation (FIG. 12, lanes 5, 7, 9 and11). In MDA-MB175, there was no significantly elevated level of erbB-3protein. Thus, in 4 out of 12 breast tumor cells lines, thegp180^(erbB-3) signaling function was activated at steady state. Whetherchronic erbB-3 phosphorylation involves autocrine stimulation or subtlestructural alterations, these findings provide evidence for constitutivegp180^(erbB-3) activation in these human breast tumor cells.

EXAMPLE 9 Identification, Purification and Characterization of erbB-3Ligands

As shown in FIG. 12, the gp180^(erbB-3) that is overexpressed in somehuman breast tumor cell lines can be either functionally activated ornot, depending on the cell line. Further, in some other human breasttumor cell lines, the erbB-3 polypeptide is not overexpressed and,again, can be either activated or not activated. These differences, andthe common property of growth factor receptor-like tyrosine kinases torapidly autophosphorylate on tyrosine residues in response to ligandtriggering, can be exploited to identify, isolate and characterizeligands, preferably specific ligands, that can activate or down-regulateerbB-3. The term “erbB-3 ligand” refers to a molecule that binds to theerbB-3 protein, particularly to the extracellular domain of the erbB-3protein, and can activate (“erbB-3 activating ligand”) or down-regulate(“erbB-3 blocking ligand”) the biochemical and/or biological activity ofthe erbB-3 protein. Depending on the concentration of the ligand, aligand can both activate and down-regulate activity.

A source containing a potential erbB-3 ligand, such as conditionedmedium, body fluid extracts, cell extracts, tissue extracts or the like,with or without agents which can modify erbB-3 activity, can be screenedfor the presence of such a ligand by the ability of the solution, in thecase of an activating ligand, to enhance erbB-3 phosphorylation. Withrespect to screening for an erbB-3 activating ligand, cells from a cellline whose expressed erbB-3 protein contains nonexistent or low levelintrinsic tyrosine phosphorylation can be contacted with potentialligand sources or control medium for a time and under conditionssufficient to allow binding of an erbB-3 ligand, if present, to bind toerbB-3. Typically, if an erbB-3 ligand is present, binding will occurwithin a short time. Thus, the cells are exposed to potential ligandsources or control medium for, preferably, no longer than 30 minutes,most preferably, 10 minutes or less. Appropriate conditions to allowbinding of the ligand can be determined by one skilled in the art, suchas physiological conditions at 37° C. or the conditions given in FIG. 5of Holmes et al., Science 256:1205 (1992). erbB-3 modifying agents, ifadministered, can be present in a concentration between 10⁻² pM and 10⁵pM. The cell line employed can overexpress erbB-3, such as the mammarytumor cell lines MDA-MB415, MDA-MB134, MDA-MB468, BT483, MDA-MB361,MCF-7 and ZR-75-1, which express an increased amount of the erbB-3protein with low level intrinsic tyrosine phosphorylation compared toprotein amounts and activation levels for corresponding nonmalignantcells. One such potential activating ligand source can be derived fromcell lines that not only overexpress erbB-3 but also exhibit high levelintrinsic tyrosine phosphorylation, such as MDA-MB453, SK-BR-3, andBT474. In addition, any normal cell which does not overexpress erbB-3can be utilized, e.g., fibroblasts.

Similarly, with respect to screening for an erbB-3 inhibitorydown-regulating ligand, a cell line whose expressed erbB-3 proteincontains high level intrinsic tyrosine phosphorylation can be exposed topotential ligand sources or control medium for a time and underconditions sufficient to allow binding of an erbB-3 ligand, if present,to bind to erbB-3. The cell line employed preferably expresses activatederbB-3, such as the mammary tumor cell lines MDA-MB453, SK-BR-3 andBT474. In addition, an activating ligand at higher concentration can bedown-regulating. Such activity can be routinely screened given theteaching herein.

The triggering or blocking of erbB-3 activation can be detected bycomparing the level of erbB-3 tyrosine phosphorylation in the cell lineafter exposure to the potential ligand source with the normal level,e.g., the level obtained after exposure to the control medium. Forexample, in a negative control the cells can be in serum free medium andfor activating ligand the conditioned medium is from cell lines withincreased erbB-3 that don't have phosphorylation. For instance, tomeasure erbB-3 specific tyrosine phosphorylation, potentially triggered(or blocked) cells and the control cells are lysed. Using proceduressuch as those discussed above, the erbB-3 protein is immunoprecipitatedwith an erbB-3 specific antibody, preferably a monoclonal such as MAbE3-1. The immunoprecipitates are divided and subjected to immunoblotanalysis with either antiphosphotyrosine or erbB-3 antibodies. Thepresence of an erbB-3 activating or blocking ligand can be monitored bya relative increase or decrease, respectively, of phosphotyrosine levelsin comparison to the untriggered control. Any increase can besignificant, especially a two-fold or greater increase. Thisligand-detection system can be used repeatedly throughout the ligandpurification procedures so as to monitor protein purification of theerbB-3 ligand to homogeneity.

Alternatively, following exposure of the cell lines to the potentialligand source as discussed above, detection of an erbB-3 activatingligand or blocking ligand can be accomplished by measurement of cellgrowth and/or mitogenic signals resulting from the activation orinhibition of erbB-3 catalytic activity, using, for example, theprocedures given in Example 7 above. An increase in colony number orcolony size and/or a dose-dependent increase of DNA synthesis for thecells exposed to the potential ligand relative to those exposed to thecontrol medium correlates with the presence of an activating ligand inthe potential ligand source. Conversely, respective decreases correlatewith the presence of a blocking ligand in the potential ligand source.

Following the isolation and purification of the erbB-3 ligand, theidentity of the ligand can be determined by protein identificationmethods known in the art, such as amino acid sequencing. Further, theerbB-3 ligand can be molecularly characterized. For instance, similar tothe procedures outlined in Holmes et al., Science 256:1205 (1992), thenucleic acid sequence that corresponds to the ligand's amino acidsequence, or a partial amino acid sequence corresponding to a portion ofthe ligand, can be used to design degenerate oligonucleotide probescorresponding to the amino acid sequence or partial sequence. Thesedegenerate oligonucleotides can be used to screen a cDNA library andgenerate a clone that encodes the precursor of the erbB-3 ligand.Following determination of the coding sequence, related coding sequencescan be discovered by screening other libraries.

For purposes of completing the background description and presentdisclosure, each of the published articles, patents and patentapplications heretofore identified in this specification are herebyincorporated by reference into the specification.

The foregoing invention has been described in some detail for purposesof clarity and understanding. It will also be obvious that variouschanges and combinations in form and detail can be made withoutdeparting from the scope of the invention.

12 1542 base pairs nucleic acid single linear DNA (genomic) exon 66..221exon 780..855 exon 1040..1185 intron 222..779 intron 856..1039 CDSjoin(66..221, 780..855, 1040..1185) 1 GAATTCCAGA TCTCAGTGAC TGATTCCCCCAACCTTAAGA ATACTTTCTT CCCCTATACC 60 TACAG GGA ATG TAC TAC CTT GAG GAACAT GGT ATG GTG CAT AGA AAC 107 Gly Met Tyr Tyr Leu Glu Glu His Gly MetVal His Arg Asn 1 5 10 CTG GCT GCC CGA AAC GTG CTA CTC AAG TCA CCC AGTCAG GTT CAG GTG 155 Leu Ala Ala Arg Asn Val Leu Leu Lys Ser Pro Ser GlnVal Gln Val 15 20 25 30 GCA GAT TTT GGT GTG GCT GAC CTG CTG CCT CCT GATGAT AAG CAG CTG 203 Ala Asp Phe Gly Val Ala Asp Leu Leu Pro Pro Asp AspLys Gln Leu 35 40 45 CTA TAC AGT GAG GCC AAG GTGAGGAGAC ACAAAGGGTAAGGAGGCGGG 251 Leu Tyr Ser Glu Ala Lys 50 GGTGGAGTGA AGCATGGGGATAGGGAGCAG CCAGTGGTCT CTTCCAGAGG CAAGCAGATG 311 CTTCATGGTA AGTTCAAGGAGAGAAGGCTG CAGATGCCAG ATATTTTAGT TCAGAGGGCA 371 ACAAAGAAAA TAATGATCAAGAACTTGGGA CTGGCCGGGC GCGGTGGCTC ACGCCTGTAA 431 TCCCAACACT TCGGGAGGCCAAGGCGGGTG GATCACAAGG TCAGGAGATC AAGACCATCC 491 TGGCTAGCAC GGTGAAACCCCGTCTCTACT AAATATACAA AAAAAAAAAA ATTAGCCAGG 551 CGTGGCGGCA TGCATCTGTACTCCCAGCTA CTCGGGAGGC TGAGGCAGGA GAATGGCGTG 611 AACCCAGGAG GCGGAGCTTGCAGTGGGCCG AGATCGCACC ACTGCACTCC AGTCTGGGCG 671 ACAGAGCGAG ACTCCGTCTCAAAAAAAAAA AAAAAAGAAT TTGGGACTTG GAAATCCTAA 731 GAAAATTTGT GGAAATAAACTTGTGATACC TCTATCTTTA ATCCGCAG ACT CCA ATT 788 Thr Pro Ile 55 AAG TGGATG GCC CTT GAG AGT ATC CAC TTT GGG AAA TAC ACA CAC CAG 836 Lys Trp MetAla Leu Glu Ser Ile His Phe Gly Lys Tyr Thr His Gln 60 65 70 AGT GAT GTCTGG AGC TAT G GTCAGTGCAT CTGGATGCCC TCTCTACCAT 885 Ser Asp Val Trp SerTyr 75 CACTGGCCCC AGTTTCAAAT TTACCTTTTG AGAGCCCCCT CTTAGAATCT CTAAGCACTT945 CAGATTTTTG TGTTAGATCA GGTTCTGCCT TCCCTTCACT TCATGCCCAT GTCTACTATT1005 TTGCCAGTGA CTAGTCCATG TCTTCCTGCA ACAG GT GTG ACA GTT TGG GAG 1056Gly Val Thr Val Trp Glu 80 TTG ATG ACC TTC GGG GCA GAG CCC TAT GCA GGGCTA CGA TTG GCT GAA 1104 Leu Met Thr Phe Gly Ala Glu Pro Tyr Ala Gly LeuArg Leu Ala Glu 85 90 95 GTA CCA GAC CTG CTA GAG AAG GGG GAG CGG TTG GCACAG CCC CAG ATC 1152 Val Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Ala GlnPro Gln Ile 100 105 110 115 TGC ACA ATT GAT GTC TAC ATG GTG ATG GTC AAGTGTGAGTTAC CTGCTGAGCC 1205 Cys Thr Ile Asp Val Tyr Met Val Met Val Lys120 125 CAACCATTTT CTCTTTTTTT CTTTTTTTTT CTTTTTTTTT TTTTTTTGAGACAGAGTCTC 1265 ACAATTGTCA CCCAGGCTGG AGTGCAATGG TGCAATCAAT CTTGGCTCACTACAACCTCC 1325 GCCTCTCGGG TTCAAGAGAT TCTCCTGCTT CAGCTCCGGA GTAGCTGGGATTACAGCGCC 1385 CGCCACACCT GGATAACTGT TACACTTTTA GTAGAGATGG GGTTTCACCATGTTGGCCAG 1445 GCTGGTCTCA AACTCCTGAC CTCAGGTGAT CCGCCTGCCT CAGCTTCCCAAAGTGCTGGG 1505 ATTACAGGTG TGAGCCATCA TGCTCGCCTG ACTGCAG 1542 126 aminoacids amino acid linear protein 2 Gly Met Tyr Tyr Leu Glu Glu His GlyMet Val His Arg Asn Leu Ala 1 5 10 15 Ala Arg Asn Val Leu Leu Lys SerPro Ser Gln Val Gln Val Ala Asp 20 25 30 Phe Gly Val Ala Asp Leu Leu ProPro Asp Asp Lys Gln Leu Leu Tyr 35 40 45 Ser Glu Ala Lys Thr Pro Ile LysTrp Met Ala Leu Glu Ser Ile His 50 55 60 Phe Gly Lys Tyr Thr His Gln SerAsp Val Trp Ser Tyr Gly Val Thr 65 70 75 80 Val Trp Glu Leu Met Thr PheGly Ala Glu Pro Tyr Ala Gly Leu Arg 85 90 95 Leu Ala Glu Val Pro Asp LeuLeu Glu Lys Gly Glu Arg Leu Ala Gln 100 105 110 Pro Gln Ile Cys Thr IleAsp Val Tyr Met Val Met Val Lys 115 120 125 4905 base pairs nucleic acidsingle linear cDNA CDS 100..4125 3 ACCAATTCGC CAGCGGTTCA GGTGGCTCTTGCCTCGATGT CCTAGCCTAG GGGCCCCCGG 60 GCCGGACTTG GCTGGGCTCC CTTCACCCTCTGCGGAGTC ATG AGG GCG AAC GAC 114 Met Arg Ala Asn Asp 1 5 GCT CTG CAGGTG CTG GGC TTG CTT TTC AGC CTG GCC CGG GGC TCC GAG 162 Ala Leu Gln ValLeu Gly Leu Leu Phe Ser Leu Ala Arg Gly Ser Glu 10 15 20 GTG GGC AAC TCTCAG GCA GTG TGT CCT GGG ACT CTG AAT GGC CTG AGT 210 Val Gly Asn Ser GlnAla Val Cys Pro Gly Thr Leu Asn Gly Leu Ser 25 30 35 GTG ACC GGC GAT GCTGAG AAC CAA TAC CAG ACA CTG TAC AAG CTC TAC 258 Val Thr Gly Asp Ala GluAsn Gln Tyr Gln Thr Leu Tyr Lys Leu Tyr 40 45 50 GAG AGG TGT GAG GTG GTGATG GGG AAC CTT GAG ATT GTG CTC ACG GGA 306 Glu Arg Cys Glu Val Val MetGly Asn Leu Glu Ile Val Leu Thr Gly 55 60 65 CAC AAT GCC GAC CTC TCC TTCCTG CAG TGG ATT CGA GAA GTG ACA GGC 354 His Asn Ala Asp Leu Ser Phe LeuGln Trp Ile Arg Glu Val Thr Gly 70 75 80 85 TAT GTC CTC GTG GCC ATG AATGAA TTC TCT ACT CTA CCA TTG CCC AAC 402 Tyr Val Leu Val Ala Met Asn GluPhe Ser Thr Leu Pro Leu Pro Asn 90 95 100 CTC CGC GTG GTG CGA GGG ACCCAG GTC TAC GAT GGG AAG TTT GCC ATC 450 Leu Arg Val Val Arg Gly Thr GlnVal Tyr Asp Gly Lys Phe Ala Ile 105 110 115 TTC GTC ATG TTG AAC TAT AACACC AAC TCC AGC CAC GCT CTG CGC CAG 498 Phe Val Met Leu Asn Tyr Asn ThrAsn Ser Ser His Ala Leu Arg Gln 120 125 130 CTC CGC TTG ACT CAG CTC ACCGAG ATT CTG TCA GGG GGT GTT TAT ATT 546 Leu Arg Leu Thr Gln Leu Thr GluIle Leu Ser Gly Gly Val Tyr Ile 135 140 145 GAG AAG AAC GAT AAG CTT TGTCAC ATG GAC ACA ATT GAC TGG AGG GAC 594 Glu Lys Asn Asp Lys Leu Cys HisMet Asp Thr Ile Asp Trp Arg Asp 150 155 160 165 ATC GTG AGG GAC CGA GATGCT GAG ATA GTG GTG AAG GAC AAT GGC AGA 642 Ile Val Arg Asp Arg Asp AlaGlu Ile Val Val Lys Asp Asn Gly Arg 170 175 180 AGC TGT CCC CCC TGT CATGAG GTT TGC AAG GGG CGA TGC TGG GGT CCT 690 Ser Cys Pro Pro Cys His GluVal Cys Lys Gly Arg Cys Trp Gly Pro 185 190 195 GGA TCA GAA GAC TGC CAGACA TTG ACC AAG ACC ATC TGT GCT CCT CAG 738 Gly Ser Glu Asp Cys Gln ThrLeu Thr Lys Thr Ile Cys Ala Pro Gln 200 205 210 TGT AAT GGT CAC TGC TTTGGG CCC AAC CCC AAC CAG TGC TGC CAT GAT 786 Cys Asn Gly His Cys Phe GlyPro Asn Pro Asn Gln Cys Cys His Asp 215 220 225 GAG TGT GCC GGG GGC TGCTCA GGC CCT CAG GAC ACA GAC TGC TTT GCC 834 Glu Cys Ala Gly Gly Cys SerGly Pro Gln Asp Thr Asp Cys Phe Ala 230 235 240 245 TGC CGG CAC TTC AATGAC AGT GGA GCC TGT GTA CCT CGC TGT CCA CAG 882 Cys Arg His Phe Asn AspSer Gly Ala Cys Val Pro Arg Cys Pro Gln 250 255 260 CCT CTT GTC TAC AACAAG CTA ACT TTC CAG CTG GAA CCC AAT CCC CAC 930 Pro Leu Val Tyr Asn LysLeu Thr Phe Gln Leu Glu Pro Asn Pro His 265 270 275 ACC AAG TAT CAG TATGGA GGA GTT TGT GTA GCC AGC TGT CCC CAT AAC 978 Thr Lys Tyr Gln Tyr GlyGly Val Cys Val Ala Ser Cys Pro His Asn 280 285 290 TTT GTG GTG GAT CAAACA TCC TGT GTC AGG GCC TGT CCT CCT GAC AAG 1026 Phe Val Val Asp Gln ThrSer Cys Val Arg Ala Cys Pro Pro Asp Lys 295 300 305 ATG GAA GTA GAT AAAAAT GGG CTC AAG ATG TGT GAG CCT TGT GGG GGA 1074 Met Glu Val Asp Lys AsnGly Leu Lys Met Cys Glu Pro Cys Gly Gly 310 315 320 325 CTA TGT CCC AAAGCC TGT GAG GGA ACA GGC TCT GGG AGC CGC TTC CAG 1122 Leu Cys Pro Lys AlaCys Glu Gly Thr Gly Ser Gly Ser Arg Phe Gln 330 335 340 ACT GTG GAC TCGAGC AAC ATT GAT GGA TTT GTG AAC TGC ACC AAG ATC 1170 Thr Val Asp Ser SerAsn Ile Asp Gly Phe Val Asn Cys Thr Lys Ile 345 350 355 CTG GGC AAC CTGGAC TTT CTG ATC ACC GGC CTC AAT GGA GAC CCC TGG 1218 Leu Gly Asn Leu AspPhe Leu Ile Thr Gly Leu Asn Gly Asp Pro Trp 360 365 370 CAC AAG ATC CCTGCC CTG GAC CCA GAG AAG CTC AAT GTC TTC CGG ACA 1266 His Lys Ile Pro AlaLeu Asp Pro Glu Lys Leu Asn Val Phe Arg Thr 375 380 385 GTA CGG GAG ATCACA GGT TAC CTG AAC ATC CAG TCC TGG CCG CCC CAC 1314 Val Arg Glu Ile ThrGly Tyr Leu Asn Ile Gln Ser Trp Pro Pro His 390 395 400 405 ATG CAC AACTTC AGT GTT TTT TCC AAT TTG ACA ACC ATT GGA GGC AGA 1362 Met His Asn PheSer Val Phe Ser Asn Leu Thr Thr Ile Gly Gly Arg 410 415 420 AGC CTC TACAAC CGG GGC TTC TCA TTG TTG ATC ATG AAG AAC TTG AAT 1410 Ser Leu Tyr AsnArg Gly Phe Ser Leu Leu Ile Met Lys Asn Leu Asn 425 430 435 GTC ACA TCTCTG GGC TTC CGA TCC CTG AAG GAA ATT AGT GCT GGG CGT 1458 Val Thr Ser LeuGly Phe Arg Ser Leu Lys Glu Ile Ser Ala Gly Arg 440 445 450 ATC TAT ATAAGT GCC AAT AGG CAG CTC TGC TAC CAC CAC TCT TTG AAC 1506 Ile Tyr Ile SerAla Asn Arg Gln Leu Cys Tyr His His Ser Leu Asn 455 460 465 TGG ACC AAGGTG CTT CGG GGG CCT ACG GAA GAG CGA CTA GAC ATC AAG 1554 Trp Thr Lys ValLeu Arg Gly Pro Thr Glu Glu Arg Leu Asp Ile Lys 470 475 480 485 CAT AATCGG CCG CGC AGA GAC TGC GTG GCA GAG GGC AAA GTG TGT GAC 1602 His Asn ArgPro Arg Arg Asp Cys Val Ala Glu Gly Lys Val Cys Asp 490 495 500 CCA CTGTGC TCC TCT GGG GGA TGC TGG GGC CCA GGC CCT GGT CAG TGC 1650 Pro Leu CysSer Ser Gly Gly Cys Trp Gly Pro Gly Pro Gly Gln Cys 505 510 515 TTG TCCTGT CGA AAT TAT AGC CGA GGA GGT GTC TGT GTG ACC CAC TGC 1698 Leu Ser CysArg Asn Tyr Ser Arg Gly Gly Val Cys Val Thr His Cys 520 525 530 AAC TTTCTG AAT GGG GAG CCT CGA GAA TTT GCC CAT GAG GCC GAA TGC 1746 Asn Phe LeuAsn Gly Glu Pro Arg Glu Phe Ala His Glu Ala Glu Cys 535 540 545 TTC TCCTGC CAC CCG GAA TGC CAA CCC ATG GAG GGC ACT GCC ACA TGC 1794 Phe Ser CysHis Pro Glu Cys Gln Pro Met Glu Gly Thr Ala Thr Cys 550 555 560 565 AATGGC TCG GGC TCT GAT ACT TGT GCT CAA TGT GCC CAT TTT CGA GAT 1842 Asn GlySer Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe Arg Asp 570 575 580 GGGCCC CAC TGT GTG AGC AGC TGC CCC CAT GGA GTC CTA GGT GCC AAG 1890 Gly ProHis Cys Val Ser Ser Cys Pro His Gly Val Leu Gly Ala Lys 585 590 595 GGCCCA ATC TAC AAG TAC CCA GAT GTT CAG AAT GAA TGT CGG CCC TGC 1938 Gly ProIle Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg Pro Cys 600 605 610 CATGAG AAC TGC ACC CAG GGG TGT AAA GGA CCA GAG CTT CAA GAC TGT 1986 His GluAsn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu Gln Asp Cys 615 620 625 TTAGGA CAA ACA CTG GTG CTG ATC GGC AAA ACC CAT CTG ACA ATG GCT 2034 Leu GlyGln Thr Leu Val Leu Ile Gly Lys Thr His Leu Thr Met Ala 630 635 640 645TTG ACA GTG ATA GCA GGA TTG GTA GTG ATT TTC ATG ATG CTG GGC GGC 2082 LeuThr Val Ile Ala Gly Leu Val Val Ile Phe Met Met Leu Gly Gly 650 655 660ACT TTT CTC TAC TGG CGT GGG CGC CGG ATT CAG AAT AAA AGG GCT ATG 2130 ThrPhe Leu Tyr Trp Arg Gly Arg Arg Ile Gln Asn Lys Arg Ala Met 665 670 675AGG CGA TAC TTG GAA CGG GGT GAG AGC ATA GAG CCT CTG GAC CCC AGT 2178 ArgArg Tyr Leu Glu Arg Gly Glu Ser Ile Glu Pro Leu Asp Pro Ser 680 685 690GAG AAG GCT AAC AAA GTC TTG GCC AGA ATC TTC AAA GAG ACA GAG CTA 2226 GluLys Ala Asn Lys Val Leu Ala Arg Ile Phe Lys Glu Thr Glu Leu 695 700 705AGG AAG CTT AAA GTG CTT GGC TCG GGT GTC TTT GGA ACT GTG CAC AAA 2274 ArgLys Leu Lys Val Leu Gly Ser Gly Val Phe Gly Thr Val His Lys 710 715 720725 GGA GTG TGG ATC CCT GAG GGT GAA TCA ATC AAG ATT CCA GTC TGC ATT 2322Gly Val Trp Ile Pro Glu Gly Glu Ser Ile Lys Ile Pro Val Cys Ile 730 735740 AAA GTC ATT GAG GAC AAG AGT GGA CGG CAG AGT TTT CAA GCT GTG ACA 2370Lys Val Ile Glu Asp Lys Ser Gly Arg Gln Ser Phe Gln Ala Val Thr 745 750755 GAT CAT ATG CTG GCC ATT GGC AGC CTG GAC CAT GCC CAC ATT GTA AGG 2418Asp His Met Leu Ala Ile Gly Ser Leu Asp His Ala His Ile Val Arg 760 765770 CTG CTG GGA CTA TGC CCA GGG TCA TCT CTG CAG CTT GTC ACT CAA TAT 2466Leu Leu Gly Leu Cys Pro Gly Ser Ser Leu Gln Leu Val Thr Gln Tyr 775 780785 TTG CCT CTG GGT TCT CTG CTG GAT CAT GTG AGA CAA CAC CGG GGG GCA 2514Leu Pro Leu Gly Ser Leu Leu Asp His Val Arg Gln His Arg Gly Ala 790 795800 805 CTG GGG CCA CAG CTG CTG CTC AAC TGG GGA GTA CAA ATT GCC AAG GGA2562 Leu Gly Pro Gln Leu Leu Leu Asn Trp Gly Val Gln Ile Ala Lys Gly 810815 820 ATG TAC TAC CTT GAG GAA CAT GGT ATG GTG CAT AGA AAC CTG GCT GCC2610 Met Tyr Tyr Leu Glu Glu His Gly Met Val His Arg Asn Leu Ala Ala 825830 835 CGA AAC GTG CTA CTC AAG TCA CCC AGT CAG GTT CAG GTG GCA GAT TTT2658 Arg Asn Val Leu Leu Lys Ser Pro Ser Gln Val Gln Val Ala Asp Phe 840845 850 GGT GTG GCT GAC CTG CTG CCT CCT GAT GAT AAG CAG CTG CTA TAC AGT2706 Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys Gln Leu Leu Tyr Ser 855860 865 GAG GCC AAG ACT CCA ATT AAG TGG ATG GCC CTT GAG AGT ATC CAC TTT2754 Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu Glu Ser Ile His Phe 870875 880 885 GGG AAA TAC ACA CAC CAG AGT GAT GTC TGG AGC TAT GGT GTG ACAGTT 2802 Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val890 895 900 TGG GAG TTG ATG ACC TTC GGG GCA GAG CCC TAT GCA GGG CTA CGATTG 2850 Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr Ala Gly Leu Arg Leu905 910 915 GCT GAA GTA CCA GAC CTG CTA GAG AAG GGG GAG CGG TTG GCA CAGCCC 2898 Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Ala Gln Pro920 925 930 CAG ATC TGC ACA ATT GAT GTC TAC ATG GTG ATG GTC AAG TGT TGGATG 2946 Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met Val Lys Cys Trp Met935 940 945 ATT GAT GAG AAC ATT CGC CCA ACC TTT AAA GAA CTA GCC AAT GAGTTC 2994 Ile Asp Glu Asn Ile Arg Pro Thr Phe Lys Glu Leu Ala Asn Glu Phe950 955 960 965 ACC AGG ATG GCC CGA GAC CCA CCA CGG TAT CTG GTC ATA AAGAGA GAG 3042 Thr Arg Met Ala Arg Asp Pro Pro Arg Tyr Leu Val Ile Lys ArgGlu 970 975 980 AGT GGG CCT GGA ATA GCC CCT GGG CCA GAG CCC CAT GGT CTGACA AAC 3090 Ser Gly Pro Gly Ile Ala Pro Gly Pro Glu Pro His Gly Leu ThrAsn 985 990 995 AAG AAG CTA GAG GAA GTA GAG CTG GAG CCA GAA CTA GAC CTAGAC CTA 3138 Lys Lys Leu Glu Glu Val Glu Leu Glu Pro Glu Leu Asp Leu AspLeu 1000 1005 1010 GAC TTG GAA GCA GAG GAG GAC AAC CTG GCA ACC ACC ACACTG GGC TCC 3186 Asp Leu Glu Ala Glu Glu Asp Asn Leu Ala Thr Thr Thr LeuGly Ser 1015 1020 1025 GCC CTC AGC CTA CCA GTT GGA ACA CTT AAT CGG CCACGT GGG AGC CAG 3234 Ala Leu Ser Leu Pro Val Gly Thr Leu Asn Arg Pro ArgGly Ser Gln 1030 1035 1040 1045 AGC CTT TTA AGT CCA TCA TCT GGA TAC ATGCCC ATG AAC CAG GGT AAT 3282 Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met ProMet Asn Gln Gly Asn 1050 1055 1060 CTT GGG GAG TCT TGC CAG GAG TCT GCAGTT TCT GGG AGC AGT GAA CGG 3330 Leu Gly Glu Ser Cys Gln Glu Ser Ala ValSer Gly Ser Ser Glu Arg 1065 1070 1075 TGC CCC CGT CCA GTC TCT CTA CACCCA ATG CCA CGG GGA TGC CTG GCA 3378 Cys Pro Arg Pro Val Ser Leu His ProMet Pro Arg Gly Cys Leu Ala 1080 1085 1090 TCA GAG TCA TCA GAG GGG CATGTA ACA GGC TCT GAG GCT GAG CTC CAG 3426 Ser Glu Ser Ser Glu Gly His ValThr Gly Ser Glu Ala Glu Leu Gln 1095 1100 1105 GAG AAA GTG TCA ATG TGTAGA AGC CGG AGC AGG AGC CGG AGC CCA CGG 3474 Glu Lys Val Ser Met Cys ArgSer Arg Ser Arg Ser Arg Ser Pro Arg 1110 1115 1120 1125 CCA CGC GGA GATAGC GCC TAC CAT TCC CAG CGC CAC AGT CTG CTG ACT 3522 Pro Arg Gly Asp SerAla Tyr His Ser Gln Arg His Ser Leu Leu Thr 1130 1135 1140 CCT GTT ACCCCA CTC TCC CCA CCC GGG TTA GAG GAA GAG GAT GTC AAC 3570 Pro Val Thr ProLeu Ser Pro Pro Gly Leu Glu Glu Glu Asp Val Asn 1145 1150 1155 GGT TATGTC ATG CCA GAT ACA CAC CTC AAA GGT ACT CCC TCC TCC CGG 3618 Gly Tyr ValMet Pro Asp Thr His Leu Lys Gly Thr Pro Ser Ser Arg 1160 1165 1170 GAAGGC ACC CTT TCT TCA GTG GGT CTT AGT TCT GTC CTG GGT ACT GAA 3666 Glu GlyThr Leu Ser Ser Val Gly Leu Ser Ser Val Leu Gly Thr Glu 1175 1180 1185GAA GAA GAT GAA GAT GAG GAG TAT GAA TAC ATG AAC CGG AGG AGA AGG 3714 GluGlu Asp Glu Asp Glu Glu Tyr Glu Tyr Met Asn Arg Arg Arg Arg 1190 11951200 1205 CAC AGT CCA CCT CAT CCC CCT AGG CCA AGT TCC CTT GAG GAG CTGGGT 3762 His Ser Pro Pro His Pro Pro Arg Pro Ser Ser Leu Glu Glu Leu Gly1210 1215 1220 TAT GAG TAC ATG GAT GTG GGG TCA GAC CTC AGT GCC TCT CTGGGC AGC 3810 Tyr Glu Tyr Met Asp Val Gly Ser Asp Leu Ser Ala Ser Leu GlySer 1225 1230 1235 ACA CAG AGT TGC CCA CTC CAC CCT GTA CCC ATC ATG CCCACT GCA GGC 3858 Thr Gln Ser Cys Pro Leu His Pro Val Pro Ile Met Pro ThrAla Gly 1240 1245 1250 ACA ACT CCA GAT GAA GAC TAT GAA TAT ATG AAT CGGCAA CGA GAT GGA 3906 Thr Thr Pro Asp Glu Asp Tyr Glu Tyr Met Asn Arg GlnArg Asp Gly 1255 1260 1265 GGT GGT CCT GGG GGT GAT TAT GCA GCC ATG GGGGCC TGC CCA GCA TCT 3954 Gly Gly Pro Gly Gly Asp Tyr Ala Ala Met Gly AlaCys Pro Ala Ser 1270 1275 1280 1285 GAG CAA GGG TAT GAA GAG ATG AGA GCTTTT CAG GGG CCT GGA CAT CAG 4002 Glu Gln Gly Tyr Glu Glu Met Arg Ala PheGln Gly Pro Gly His Gln 1290 1295 1300 GCC CCC CAT GTC CAT TAT GCC CGCCTA AAA ACT CTA CGT AGC TTA GAG 4050 Ala Pro His Val His Tyr Ala Arg LeuLys Thr Leu Arg Ser Leu Glu 1305 1310 1315 GCT ACA GAC TCT GCC TTT GATAAC CCT GAT TAC TGG CAT AGC AGG CTT 4098 Ala Thr Asp Ser Ala Phe Asp AsnPro Asp Tyr Trp His Ser Arg Leu 1320 1325 1330 TTC CCC AAG GCT AAT GCCCAG AGA ACG TAACTCCTGC TCCCTGTGGC 4145 Phe Pro Lys Ala Asn Ala Gln ArgThr 1335 1340 ACTCAGGGAG CATTTAATGG CAGCTAGTGC CTTTAGAGGG TACCGTCTTCTCCCTATTCC 4205 CTCTCTCTCC CAGGTCCCAG CCCCTTTTCC CCAGTCCCAG ACAATTCCATTCAATCTTTG 4265 GAGGCTTTTA AACATTTTGA CACAAAATTC TTATGGTATG TAGCCAGCTGTGCACTTTCT 4325 TCTCTTTCCC AACCCCAGGA AAGGTTTTCC TTATTTTGTG TGCTTTCCCAGTCCCATTCC 4385 TCAGCTTCTT CACAGGCACT CCTGGAGATA TGAAGGATTA CTCTCCATATCCCTTCCTCT 4445 CAGGCTCTTG ACTACTTGGA ACTAGGCTCT TATGTGTGCC TTTGTTTCCCATCAGACTGT 4505 CAAGAAGAGG AAAGGGAGGA AACCTAGCAG AGGAAAGTGT AATTTTGGTTTATGACTCTT 4565 AACCCCCTAG AAAGACAGAA GCTTAAAATC TGTGAAGAAA GAGGTTAGGAGTAGATATTG 4625 ATTACTATCA TAATTCAGCA CTTAACTATG AGCCAGGCAT CATACTAAACTTCACCTACA 4685 TTATCTCACT TAGTCCTTTA TCATCCTTAA AACAATTCTG TGACATACATATTATCTCAT 4745 TTTACACAAA GGGAAGTCGG GCATGGTGGC TCATGCCTGT AATCTCAGCACTTTGGGAGG 4805 CTGAGGCAGA AGGATTACCT GAGGCAAGGA GTTTGAGACC AGCTTAGCCAACATAGTAAG 4865 ACCCCCATCT CTTTAAAAAA AAAAAAAAAA AAAAAAAAAA 4905 1342amino acids amino acid linear protein 4 Met Arg Ala Asn Asp Ala Leu GlnVal Leu Gly Leu Leu Phe Ser Leu 1 5 10 15 Ala Arg Gly Ser Glu Val GlyAsn Ser Gln Ala Val Cys Pro Gly Thr 20 25 30 Leu Asn Gly Leu Ser Val ThrGly Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45 Leu Tyr Lys Leu Tyr Glu ArgCys Glu Val Val Met Gly Asn Leu Glu 50 55 60 Ile Val Leu Thr Gly His AsnAla Asp Leu Ser Phe Leu Gln Trp Ile 65 70 75 80 Arg Glu Val Thr Gly TyrVal Leu Val Ala Met Asn Glu Phe Ser Thr 85 90 95 Leu Pro Leu Pro Asn LeuArg Val Val Arg Gly Thr Gln Val Tyr Asp 100 105 110 Gly Lys Phe Ala IlePhe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 115 120 125 His Ala Leu ArgGln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser 130 135 140 Gly Gly ValTyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr 145 150 155 160 IleAsp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val 165 170 175Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys Gly 180 185190 Arg Cys Trp Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu Thr Lys Thr 195200 205 Ile Cys Ala Pro Gln Cys Asn Gly His Cys Phe Gly Pro Asn Pro Asn210 215 220 Gln Cys Cys His Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro GlnAsp 225 230 235 240 Thr Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser GlyAla Cys Val 245 250 255 Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys LeuThr Phe Gln Leu 260 265 270 Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr GlyGly Val Cys Val Ala 275 280 285 Ser Cys Pro His Asn Phe Val Val Asp GlnThr Ser Cys Val Arg Ala 290 295 300 Cys Pro Pro Asp Lys Met Glu Val AspLys Asn Gly Leu Lys Met Cys 305 310 315 320 Glu Pro Cys Gly Gly Leu CysPro Lys Ala Cys Glu Gly Thr Gly Ser 325 330 335 Gly Ser Arg Phe Gln ThrVal Asp Ser Ser Asn Ile Asp Gly Phe Val 340 345 350 Asn Cys Thr Lys IleLeu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu 355 360 365 Asn Gly Asp ProTrp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu 370 375 380 Asn Val PheArg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln 385 390 395 400 SerTrp Pro Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr 405 410 415Thr Ile Gly Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser Leu Leu Ile 420 425430 Met Lys Asn Leu Asn Val Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu 435440 445 Ile Ser Ala Gly Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr450 455 460 His His Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr GluGlu 465 470 475 480 Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp CysVal Ala Glu 485 490 495 Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly GlyCys Trp Gly Pro 500 505 510 Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn TyrSer Arg Gly Gly Val 515 520 525 Cys Val Thr His Cys Asn Phe Leu Asn GlyGlu Pro Arg Glu Phe Ala 530 535 540 His Glu Ala Glu Cys Phe Ser Cys HisPro Glu Cys Gln Pro Met Glu 545 550 555 560 Gly Thr Ala Thr Cys Asn GlySer Gly Ser Asp Thr Cys Ala Gln Cys 565 570 575 Ala His Phe Arg Asp GlyPro His Cys Val Ser Ser Cys Pro His Gly 580 585 590 Val Leu Gly Ala LysGly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn 595 600 605 Glu Cys Arg ProCys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro 610 615 620 Glu Leu GlnAsp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr 625 630 635 640 HisLeu Thr Met Ala Leu Thr Val Ile Ala Gly Leu Val Val Ile Phe 645 650 655Met Met Leu Gly Gly Thr Phe Leu Tyr Trp Arg Gly Arg Arg Ile Gln 660 665670 Asn Lys Arg Ala Met Arg Arg Tyr Leu Glu Arg Gly Glu Ser Ile Glu 675680 685 Pro Leu Asp Pro Ser Glu Lys Ala Asn Lys Val Leu Ala Arg Ile Phe690 695 700 Lys Glu Thr Glu Leu Arg Lys Leu Lys Val Leu Gly Ser Gly ValPhe 705 710 715 720 Gly Thr Val His Lys Gly Val Trp Ile Pro Glu Gly GluSer Ile Lys 725 730 735 Ile Pro Val Cys Ile Lys Val Ile Glu Asp Lys SerGly Arg Gln Ser 740 745 750 Phe Gln Ala Val Thr Asp His Met Leu Ala IleGly Ser Leu Asp His 755 760 765 Ala His Ile Val Arg Leu Leu Gly Leu CysPro Gly Ser Ser Leu Gln 770 775 780 Leu Val Thr Gln Tyr Leu Pro Leu GlySer Leu Leu Asp His Val Arg 785 790 795 800 Gln His Arg Gly Ala Leu GlyPro Gln Leu Leu Leu Asn Trp Gly Val 805 810 815 Gln Ile Ala Lys Gly MetTyr Tyr Leu Glu Glu His Gly Met Val His 820 825 830 Arg Asn Leu Ala AlaArg Asn Val Leu Leu Lys Ser Pro Ser Gln Val 835 840 845 Gln Val Ala AspPhe Gly Val Ala Asp Leu Leu Pro Pro Asp Asp Lys 850 855 860 Gln Leu LeuTyr Ser Glu Ala Lys Thr Pro Ile Lys Trp Met Ala Leu 865 870 875 880 GluSer Ile His Phe Gly Lys Tyr Thr His Gln Ser Asp Val Trp Ser 885 890 895Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ala Glu Pro Tyr 900 905910 Ala Gly Leu Arg Leu Ala Glu Val Pro Asp Leu Leu Glu Lys Gly Glu 915920 925 Arg Leu Ala Gln Pro Gln Ile Cys Thr Ile Asp Val Tyr Met Val Met930 935 940 Val Lys Cys Trp Met Ile Asp Glu Asn Ile Arg Pro Thr Phe LysGlu 945 950 955 960 Leu Ala Asn Glu Phe Thr Arg Met Ala Arg Asp Pro ProArg Tyr Leu 965 970 975 Val Ile Lys Arg Glu Ser Gly Pro Gly Ile Ala ProGly Pro Glu Pro 980 985 990 His Gly Leu Thr Asn Lys Lys Leu Glu Glu ValGlu Leu Glu Pro Glu 995 1000 1005 Leu Asp Leu Asp Leu Asp Leu Glu AlaGlu Glu Asp Asn Leu Ala Thr 1010 1015 1020 Thr Thr Leu Gly Ser Ala LeuSer Leu Pro Val Gly Thr Leu Asn Arg 1025 1030 1035 1040 Pro Arg Gly SerGln Ser Leu Leu Ser Pro Ser Ser Gly Tyr Met Pro 1045 1050 1055 Met AsnGln Gly Asn Leu Gly Glu Ser Cys Gln Glu Ser Ala Val Ser 1060 1065 1070Gly Ser Ser Glu Arg Cys Pro Arg Pro Val Ser Leu His Pro Met Pro 10751080 1085 Arg Gly Cys Leu Ala Ser Glu Ser Ser Glu Gly His Val Thr GlySer 1090 1095 1100 Glu Ala Glu Leu Gln Glu Lys Val Ser Met Cys Arg SerArg Ser Arg 1105 1110 1115 1120 Ser Arg Ser Pro Arg Pro Arg Gly Asp SerAla Tyr His Ser Gln Arg 1125 1130 1135 His Ser Leu Leu Thr Pro Val ThrPro Leu Ser Pro Pro Gly Leu Glu 1140 1145 1150 Glu Glu Asp Val Asn GlyTyr Val Met Pro Asp Thr His Leu Lys Gly 1155 1160 1165 Thr Pro Ser SerArg Glu Gly Thr Leu Ser Ser Val Gly Leu Ser Ser 1170 1175 1180 Val LeuGly Thr Glu Glu Glu Asp Glu Asp Glu Glu Tyr Glu Tyr Met 1185 1190 11951200 Asn Arg Arg Arg Arg His Ser Pro Pro His Pro Pro Arg Pro Ser Ser1205 1210 1215 Leu Glu Glu Leu Gly Tyr Glu Tyr Met Asp Val Gly Ser AspLeu Ser 1220 1225 1230 Ala Ser Leu Gly Ser Thr Gln Ser Cys Pro Leu HisPro Val Pro Ile 1235 1240 1245 Met Pro Thr Ala Gly Thr Thr Pro Asp GluAsp Tyr Glu Tyr Met Asn 1250 1255 1260 Arg Gln Arg Asp Gly Gly Gly ProGly Gly Asp Tyr Ala Ala Met Gly 1265 1270 1275 1280 Ala Cys Pro Ala SerGlu Gln Gly Tyr Glu Glu Met Arg Ala Phe Gln 1285 1290 1295 Gly Pro GlyHis Gln Ala Pro His Val His Tyr Ala Arg Leu Lys Thr 1300 1305 1310 LeuArg Ser Leu Glu Ala Thr Asp Ser Ala Phe Asp Asn Pro Asp Tyr 1315 13201325 Trp His Ser Arg Leu Phe Pro Lys Ala Asn Ala Gln Arg Thr 1330 13351340 15 amino acids amino acid linear peptide internal 5 Glu Asp Glu AspGlu Glu Tyr Glu Tyr Met Asn Arg Arg Arg Arg 1 5 10 15 15 amino acidsamino acid linear peptide internal 6 Thr Thr Pro Asp Glu Asp Tyr Glu TyrMet Asn Arg Gln Arg Asp 1 5 10 15 15 amino acids amino acid linearpeptide internal 7 Thr Glu Glu Arg Leu Asp Ile Lys His Asn Arg Pro ArgArg Asp 1 5 10 15 15 amino acids amino acid linear peptide internal 8Arg Ser Arg Ser Arg Ser Arg Ser Pro Arg Pro Arg Gly Asp Ser 1 5 10 15 15amino acids amino acid linear peptide internal 9 Tyr Met Asn Arg Arg ArgArg His Ser Pro Pro His Pro Pro Arg 1 5 10 15 14 base pairs nucleic acidsingle linear DNA (genomic) 10 GATCTCGAGT CGAC 14 14 base pairs nucleicacid single linear DNA (genomic) 11 GATCGTCGAC TCGA 14 5 amino acidsamino acid linear peptide internal 12 Tyr Glu Tyr Met Asn 1 5

What is claimed is:
 1. An isolated nucleic acid which specificallyhybridizes under high stringency conditions to an erbB-3 nucleic acidconsisting of the nucleotide sequence set forth in SEQ ID NO:3 or to afragment thereof, wherein the isolated nucleic acid does not hybridizeunder high stringency conditions to an erbB-2 nucleic acid or to anepidermal growth factor receptor nucleic acid.
 2. A recombinant nucleicacid comprising the nucleic acid of claim 1 and a vector.
 3. A cellcomprising the recombinant nucleic acid of claim
 2. 4. A composition ofmatter comprising the isolated nucleic acid of claim 1, in a carrier. 5.An isolated nucleic acid fully complementary to and of the same lengthas the isolated nucleic acid of claim
 1. 6. A composition of mattercomprising the isolated nucleic acid of claim 5 in a carrier.
 7. Anisolated nucleic acid which specifically hybridizes under highstringency conditions to an erbB-3 gene sequence consisting of thenucleotide sequence of SEQ ID NO:1 or to a fragment thereof, wherein theisolated nucleic acid does not hybridize under high stringencyconditions to an erbB-2 gene sequence or to an epidermal growth factorreceptor gene sequence.
 8. A recombinant nucleic acid comprising thenucleic acid of claim 7 and a vector.
 9. A cell comprising therecombinant nucleic acid of claim
 8. 10. A composition of mattercomprising the isolated nucleic acid of claim 7, in a carrier.
 11. Anisolated nucleic acid fully complementary to and of the same length asthe isolated nucleic acid of claim
 7. 12. A composition of mattercomprising the isolated nucleic acid of claim 11 in a carrier.
 13. AcDNA that encodes SEQ ID NO:4.
 14. A recombinant nucleic acid comprisingthe cDNA of claim 13 and a vector.
 15. A cell comprising the recombinantnucleic acid of claim
 14. 16. The cDNA of claim 13, wherein the cDNAcomprises the nucleotide sequence set forth in SEQ ID NO:
 3. 17. Anisolated nucleic acid which specifically hybridizes under highstringency conditions to a genomic fragment of the erbB-3 gene or to afragment thereof, wherein the genomic fragment of the erbB-3 geneconsists of a 9 kb SacI restriction fragment of genomic DNA comprisingthe restriction map shown in FIG. 2 and the nucleotide sequence setforth in SEQ ID NO: 1, wherein the isolated nucleic acid does nothybridize under high stringency conditions to an erbB-2 gene sequence orto an epidermal growth factor receptor gene sequence.
 18. A method ofdetecting the presence of nucleic acid encoding erbB-3 in a biologicalsample, comprising: a) contacting the biological sample with the nucleicacid of claim 1 under conditions whereby hybridization of the nucleicacid to nucleic acid encoding erbB-3 can occur; and b) detectinghybridization, whereby the detection of hybridization indicates thepresence of nucleic acid encoding erbB-3 in the biological sample. 19.The method of claim 18, wherein the nucleic acid encoding erbB-3 ismessenger RNA.
 20. The method of claim 18, wherein the nucleic acidencoding erbB-3 is cDNA.
 21. The method of claim 18, wherein the nucleicacid encoding erbB-3 is genomic DNA.
 22. A method of detecting thepresence of nucleic acid encoding erbB-3 in a biological sample,comprising: a) contacting the biological sample with the isolatednucleic acid of claim 7 under conditions whereby hybridization of theisolated nucleic acid to nucleic acid encoding erbB-3 can occur; and b)detecting hybridization, whereby the detection of hybridizationindicates the presence of nucleic acid encoding erbB-3 in the biologicalsample.
 23. The method of claim 22, wherein the nucleic acid encodingerbB-3 is messenger RNA.
 24. The method of claim 22, wherein the nucleicacid encoding erbB-3 is DNA.
 25. The method of claim 22, wherein thenucleic acid encoding erbB-3 is genomic DNA.
 26. A method of detectingthe presence of nucleic acid encoding erbB-3 in a biological sample,comprising: a) contacting the biological sample with the nucleic acid ofclaim 17 under conditions whereby hybridization of the nucleic acid tonucleic acid encoding erbB-3 can occur; and b) detecting hybridization,whereby the detection of hybridization indicates the presence of nucleicacid encoding erbB-3 in the biological sample.
 27. The method of claim26, wherein the nucleic acid encoding erbB-3 is messenger RNA.
 28. Themethod of claim 26, wherein the nucleic acid encoding erbB-3 is cDNA.29. The method of claim 26, wherein the nucleic acid encoding erbB-3 isgenomic DNA.